Methods for the in planta transformation of plants and manufacturing processes and products based and obtainable thereform

ABSTRACT

The present invention relates to in planta transformation methods for plants or plant materials with a genetic construct, wherein at least one meristematic cell of a meristematic tissue of an immature inflorescence able to differentiate into a gamete of a pollen or of an ovule is exposed and then transformed, wherein the transformation can be performed to yield a stable integration or to yield a transient introduction of a genetic construct of interest. Further provided are methods for manufacturing a transgenic plant, or methods for manufacturing a genetically manipulated plant based on the in planta transformation methods according to the present invention. In addition, there is provided a plant or a progeny thereof manufactured according to the transformation and/or manufacturing methods according to the present invention.

TECHNICAL FIELD

The present invention relates to in planta transformation methods for plants or plant materials with a genetic construct, wherein at least one meristematic cell of a meristematic tissue of an immature inflorescence able to differentiate into a gamete of a pollen or of an ovule is exposed and then transformed, wherein the transformation can be performed to yield a stable integration or to yield a transient introduction of a genetic construct of interest. Further provided are methods for manufacturing a transgenic plant, or methods for manufacturing a genetically manipulated plant based on the in planta transformation methods according to the present invention. In addition, there is provided a plant or a progeny thereof manufactured according to the transformation and/or manufacturing methods according to the present invention. Finally, there is provided the use of the inventive methods and plants for the manufacture of a transgenic or genetically manipulated plant or plant material.

BACKGROUND OF THE INVENTION

Over the last decades many techniques for the genetic transformation of plants have been developed. All said methods have the ultimate goal to obtain a transgenic plant containing in all or part of the cells a foreign nucleic acid comprising a gene or a feature of interest, in the case of a gene a transgene, but with the rise of new genome engineering techniques, for example the CRISPR/Cas technology (see e.g. U.S. Pat. No. 8,697,359 B1, EP 2 800 811 A1 or WO 2013/142578 A1) also the insertion of ribonucleic acids or sequences encoding the same and of amino acid sequences becomes more and more relevant. In the case of nucleic acid molecules encoding a transgene optionally further elements, the stable integration into a plants' genome, particularly the nuclear genome, but also into the genome of e.g. plant plastids, is one of the major aims of the preceding transformation process to yield a stable “knock-in”, but likewise a stably inheritable “knock-out” of a gene or region of interest. For other applications, for instance when designing experiments using genome editing tools like CRISPR/Cas or CRISPR/Cpf1, the transient expression of the construct of interest to be transformed or transfected might be of interest to just temporarily influence the genetic material of a plant, i.e. the genome, or the transcriptome, i.e. all RNA material, thereof in a controlled way.

CRISPR system means that a small individual suitable non-coding RNA in combination with a Cas nuclease or another CRISPR nuclease like a Cpf1 nuclease (Zetsche et al., “Cpf1 Is a Singel RNA-Guides Endonuclease of a Class 2 CRISPR-Cas System”, Cell, 163, pp. 1-13, Oktober 2015) can produce a specific DNA double-stranded break in a natural environment. In artificial CRISPR systems, a modified CRISPR nuclease, modified to act as nickase or lacking any nuclease function, can be used in combination with at least one artificial guide RNA or gRNA combining the function of a crRNA and/or a tracrRNA (Makarova et al., Nature Rev. Microbiol., 2015). The immune response mediated by CRISPR/Cas in natural systems requires CRISPR-RNA (crRNA), wherein the maturation of this guiding RNA, which controls the specific activation of the CRISPR nuclease, varies significantly between the various CRISPR systems which have been characterized so far. Firstly, the invading DNA, also known as a spacer, is integrated between two adjacent repeat regions at the proximal end of the CRISPR locus. Type II CRISPR systems code for a Cas9 nuclease as key enzyme for the interference step, which system contains both a crRNA and also a trans-activating RNA (tracrRNA) as the guide motif. These hybridize and form double-stranded (ds) RNA regions which are recognized by RNAseIII and can be cleaved in order to form mature crRNAs. These then in turn associate with the Cas molecule in order to direct the nuclease specifically to the target nucleic acid region. Recombinant gRNA molecules can comprise both the variable DNA recognition region and also the Cas interaction region and thus can be specifically designed, independently of the specific target nucleic acid and the desired Cas nuclease. As a further safety mechanism, PAMs (protospacer adjacent motifs) must be present in the target nucleic acid region; these are DNA sequences which follow on directly from the Cas9/RNA complex-recognized DNA. The PAM sequence for the Cas9 from Streptococcus pyogenes has been described to be “NGG” or “NAG” (Standard IUPAC nucleotide code) (Jinek et al, “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity”, Science 2012, 337: 816-821). The PAM sequence for Cas9 from Staphylococcus aureus is “NNGRRT” or “NNGRR(N)”. Further variant CRISPR/Cas9 systems are known. Thus, a Neisseria meningitidis Cas9 cleaves at the PAM sequence NNNNGATT. A Streptococcus thermophilus Cas9 cleaves at the PAM sequence NNAGAAW. Recently, a further PAM motif NNNNRYAC has been described for a CRISPR system of Campylobacter (WO 2016/021973 A1). For Cpf1 nucleases it has been described that the Cpf1-crRNA complex efficiently cleave target DNA proceeded by a short T-rich PAM in contrast to the commonly G-rich PAMs recognized by Cas9 systems (Zetsche et al., “Cpf1 Is a Singel RNA-Guides Endonuclease of a Class 2 CRISPR-Cas System”, Cell, 163, pp. 1-13, October 2015).

Furthermore, by using modified Cas polypeptides, specific single-stranded breaks can be obtained. The combined use of Cas nickases with various recombinant gRNAs can also induce highly specific DNA double-stranded breaks by means of double DNA nicking. By using two gRNAs, moreover, the specificity of the DNA binding and thus the DNA cleavage can be optimized.

Currently, there exists a variety of plant transformation methods to introduce genetic material in the form of a genetic construct into a plant cell of interest. One preferred technique is transformation with Agrobacterium spp. which has been used for decades for a variety of different plant materials. Agrobacterium transformation, however, is much better established for dicotyledonous plants than for monocotyledonous plants, the latter comprising economically important plants e.g. in the family of Poacea/Graminaceae including inter alia plants like maize/corn, wheat, barley, rye, sorghum or sugar cane. Over the recent years, some progress could be achieved for other plant transformation methods by choosing direct delivery techniques for introducing genetic material into a plant cell, e.g. by choosing direct delivery techniques ranging from polyethylene glycol (PEG) treatment of protoplasts (Potrykus et al. 1985), procedures like electroporation (D′Halluin et al., 1992), microinjection (Neuhaus et al., 1987),silicon carbide fiber whisker technology (Kaeppler et al., 1992), viral vector mediated approaches (Gelvin, Nature Biotechnology 23, “Viral-mediated plant transformation gets a boost”, 684-685 (2005)) and particle bombardment (see e.g. Sood et al., 2011, Biologia Plantarum, 55, 1-15). For all the above methods, it is still mandatory to have an explanted plant material, including embryogenic calli or suspension cells, scutellar tissue, calli of different types, including type 1 and type 2 and organogenic type 3 calli, protoplasts, immature embryos, or green tissue which is then treated in vitro, i.e. ex planta, and not within the living plant.

One exemplary transformation process for corn is Agrobacterium mediated transformation of immature embryos of defined genotypes through somatic embryogenesis. The process of regenerating a plant from the in vitro transformed material is, however, time consuming, expensive and the efficiency and the parameters to follow are strongly genotype dependent. In addition, only few genotypes are suitable for genetic transformation, requiring backcrosses of the generated transgenic material with elite varieties which is time consuming and costly.

U.S. Pat. No. 6,603,061 B1 discloses method of transforming a corn plant cell or plant tissue using an Agrobacterium mediated process by inoculating a transformable plant cell or tissue from a corn plant with Agrobacterium containing at least one genetic component capable of being transferred to the plant cell or tissue in an inoculation media containing an effective amount of at least one antibiotic that inhibits or suppresses the growth of Agrobacterium. There are several publications in the scientific literature about Agrobacterium mediated transformation of flowers like the floral dip methodologies, routinely applied to Arabidopsis, but also to crops like wheat (Clough and Bent 1998; Zale et al. 2009). Said approaches, however, intrinsically require the cumbersome regeneration of the transformed plant cell or tissue to obtain a transgenic or genetically modified plant.

Pace and Dupuis (“Gene transfer to maize male reproductive structure by particle bombardment”, 1993, Plant Cell reports, 12: 607-611.) discloses a method for transforming tassel primordia of maize using particle bombardment for transient expression of foreign genes. The method according to Pace and Dupuis relies on the bombardment in vitro and all experiments are performed with explanted cells and no reproductive plant or plant material was or can be obtained from this approach. Pareddy and Greyson (1985) published a method wherein maize tassels (1 month old) of the genotype OH43 were dissected and bombarded in tassel maturation media. The bombardment was done at 1350 psi with 1.6 μm gold particles and the tassels were bombarded 4 times. The tassels cultured in vitro matured, but produced a low amount of anthers comparing to the greenhouse plants. Transient expression in the tassels was assayed. Most of it was detected in the outer glumes, only some in the stamen primordia. After 1 month of in vitro culture anthers were produced and tested for gus activity. Only 0.5% of all anthers showed gus activity, mostly in the vascular tissue of the anther. No analyses were done on pollen and no pollinations were performed. Therefore, there is still need for improving this method to obtain sufficient amount of transformed cells having a high transformation rate.

In wheat, a bombardment technology called “micro-targeting” was used to transform isolated immature spikes of wheat (Leduc et al. 1994). Those spikes were cultured in osmoticum medium to increase the transient expression. Even though around 80% of the particles were targeted to the L1 layer of cells (the outer layer that will produce all the epidermis tissue of the ear) and approximately 20% targeted L2 cells (that will produce reproductive organs). The authors obtained “multicellular sectors showing transgene expression” 12 days after bombardment. Those sectors were found in primordia of vegetative and reproductive organs like anthers. They describe this methodology as a possible “direct transformation of sporogenic tissues”. However, there is no follow-up of the technology, especially no follow up in planta or for other important crop plants.

The bombardment of plant cells with a nucleic acid of interest represents another process for transformation and is known since the late 1980′s (Taylor and Fauquet, 2002) for manipulating the genome of plants (at that time) recalcitrant to transformation via Agrobacterium, including inter alia cereals (Klein et al 1987; Taylor and Fauquet, 2002). The transformation via particle bombardment uses a microprojectile of metal covered with the gene of interest, which is then shot onto the target cells using an equipment known as “gene gun” (Sandford et al. 1987) at high velocity fast enough (˜1500 km/h) to penetrate the cell wall of a target tissue, but not harsh enough to cause cell death. The precipitated nucleic acid or the nucleic acid construct on the at least one microprojectile is released into the cell after bombardment, and integrated into the genome. The acceleration of microprojectiles is accomplished by a high voltage electrical discharge or compressed gas (helium). This technique was progressively further developed, e.g. for inserting more than one gene of interest into target cells (Chen et al. 1998; Wu et al. 2002). Still, several physical parameters correlated with the biolistic equipment such as pressure, distance of the macro and micro-carrier flight, and vacuum, must be optimized for a successful transformation. Another crucial parameter is the target cell or tissue itself, as this target site has to be readily accessible, as well as the nucleic acid construct of interest to be introduced.

Despite transformation methods based on biological approaches, like Agrobacterium transformation or viral vector mediated plant transformation, and methods based on physical delivery methods, like particle bombardment or microinjection, have evolved as prominent techniques for introducing genetic material into a plant cell or tissue of interest over the last years, there are still several problems associated therewith hampering the broad approach for both transient as well as stable introduction of a construct of interest in any kind of plant to be transformed.

Helenius et al. (“Gene delivery into intact plants using the Helios™ Gene Gun”, Plant Molecular Biology Reporter, 2000, 18 (3):287-288) discloses a particle bombardment as physical method for transient transformation of gene constructs into intact plant tissue of model plants like Arabidposis and tobacco. There are, however, no examples concerning either the achievement of stable transformation of the genetic construct or any advice of how to use the technology for major crop plants in plant organs other than leaves. Furthermore, the document is silent as to a method for directly obtaining a genetically modified seed directly in a modified plant obviating the need for in vitro cultivation or further crossing steps, as the target tissue is not a meristematic tissue.

Thus, it would represent a preferable targeting strategy to specifically transform the meristematic tissue of a plant of interest, as a targeted genetic manipulation effected for at least one meristematic cell provides the advantage of inheriting said manipulation to the progeny of said not yet fully differentiated cells. To this end, however, a meristematic target structure has to be exposed in a suitable way to avoid the destruction of the plant organism and/or the target structure of interest. As elucidated in FIGS. 2 and 3, the meristem in seedlings and older maize plants is completely surrounded by leaves so that the meristematic tissue has to be prepared before exposing it to a further treatment. The principle of providing a meristematic plant tissue as target structure is disclosed in DE 10 2015 006 335.9 and DE 10 2015 014 252.6.

The tissues “in planta” that can develop reproductive organs are limited. The main one is the shoot meristem. The shoot meristem is the group of cells that will divide to produce all the vegetative and reproductive organs above ground. It consists of a limited number of cells that can be programmed to differentiate in all the organs of the plant. This meristem usually has a dome shape. The outer line of cells is called L1 layer and from this, all the epidermic tissues will be formed. The inner layers of the meristem (L2 and L3) will generate the rest of the organs and those are the ones of special interest for targeting according to the present disclosure. The meristem is formed very early in embryo development. After plant vegetative growth, the meristem develops into a flower meristem that will generate the reproductive organs of the plants. In conclusion, the tissues that can be targeted “in planta” to produce modified reproductive organs are: (1) the shoot meristem in the embryo, (2) the shoot meristem in plantlets or in planta during vegetative stage and (3) the flower meristem or flower primordia. A targeting of said meristematic cells and tissues with a genome editing strategy of choice in a transient and stable way is thus of great interest, particularly as a transformation method that would be genotype and in vitro culture independent, i.e. which could be performed directly in planta in a plant of interest, would thus be desirable to save time and resources. The possibility of adapting the transformation process to a variety of monocot and dicot plants of economic interest would be highly beneficial. Furthermore, there is a need in providing plant transformation methods, which are useful for both the transient and the stable introduction of a construct of interest in a plant. Finally, it remains an outstanding need to create stable or transient transformation of a plant cell or a plant material in planta which directly yields transformed meristematic structures in a plant within an inflorescence meristematic cell or tissue, as this would pave the way for the direct acquisition of plant reproductive material directly in the modified plant and not only from the progeny thereof or after cumbersome and expensive in vitro cultivation.

SUMMARY OF THE INVENTION

The primary object of the present invention was thus the provision of methods for the genotype independent in planta transformation of a plant or a plant material, which is suitable for both, the transient as well as the stable introduction/integration of a genetic construct of interest into at least one meristematic cell of a meristematic tissue belonging to the immature inflorescence of a plant. Furthermore, it was an object to provide methods for the manufacturing of a transgenic or a genetically manipulated plant based on an in planta approach for transforming a plant or plant material. Finally it was an object to obtain a plant or a progeny of a plant manufactured by the methods according to the present invention or plant cells or a plant material, or derivatives thereof or to provide and use a plant transformed by the methods according to the present invention for the manufacture of a transgenic or genetically manipulated plant or plant material. Finally, it was an object to define meristem access and meristem delivery methods suitable for a variety of different target plants, and to define genetic constructs and delivery methods to provide a versatile and efficient genome editing approach to modify a variety of relevant crop plants in a targeted and efficient way, either transiently or in a stable way, where all methods can be performed completely in planta obviating the need for cumbersome in vitro cultures.

The primary object has been achieved by providing in one aspect a method for the in planta transformation of a plant or plant material with a genetic construct of interest comprising the following steps: (i) providing a plant comprising at least one meristematic cell of a meristematic tissue of an immature inflorescence, wherein the meristematic cell is able to differentiate into a gamete of a pollen or of an ovule; (ii) exposing the at least one meristematic cell of the meristematic tissue of the immature inflorescence; (iii) providing a genetic construct of interest; and (iv) transforming the at least one meristematic cell of the meristematic tissue of the immature inflorescence by introducing the genetic construct of interest under suitable conditions to allow the functional integration of the genetic construct of interest into the at least one meristematic cell of the meristematic tissue of the immature inflorescence.

In one embodiment, the transformation is performed in vitro culture free.

In another embodiment, the functional integration of the genetic construct of interest into the at least one meristematic cell of the meristematic tissue of the immature inflorescence of step (iv) of the above aspect is performed as (a) a stable integration so that the integrated genetic construct of interest, or a part thereof, is heritable to a progeny, preferably whereby a transformed gamete of the pollen or of the ovule is generated from the at least one transformed meristematic cell and the transformed gamete of the pollen or of the ovule is fused to a zygote from which the progeny comprising the genetic construct of interest is developed; as transgene, or (b) a transient introduction allowing a targeted genetic manipulation of the at least one meristematic cell of the meristematic tissue of the immature inflorescence through the genetic construct of interest or products thereof, wherein the targeted genetic manipulation but not the genetic construct of interest, or a part thereof, is heritable to a progeny, preferably whereby a genetic manipulated gamete of the pollen or of the ovule is generated from the transformed at least one meristematic cell and the genetic manipulated gamete of the pollen or of the ovule is fused to a zygote from which the progeny comprising the targeted genetic manipulation is developed.

In another embodiment of the above aspect, the method comprises an additional step (v) comprising determining the functional integration of the genetic construct of interest.

In yet another embodiment of the above aspect according to the present invention, the introduction of the genetic construct of interest according to step (iv) of the above aspect is conducted using a means selected from the group consisting of a device suitable for particle bombardment, including a gene gun, transformation, including transformation using Agrobacterium spp. or using a viral vector, microinjection, electroporation, whisker technology, including silicon carbide whisker technology, and transfection, or a combination thereof.

There is provided a further embodiment, wherein step (ii) of the above aspect comprises the production of an artificially introduced window in the region, where the meristematic tissue of the immature inflorescence is located, preferably in the shoot axis, in particular in the axil, halm, culm or stem of the plant.

In yet another embodiment this aspect according to the present invention the plant or the plant material is or is part of a monocotyledonous (monocot) plant, or is or is part of a dicotyledonous (dicot) plant.

In a further embodiment according to the above aspect according to the present invention the plant or the plant material is or is part of a plant from a species selected from the group consisting of Hordeum vulgare, Hordeum bulbusom, Sorghum bicolor, Saccharum officinarium, Zea mays, Setaria italica, Oryza minuta, Oryza sativa, Oryza australiensis, Oryza alta, Triticum aestivum, Triticale, Secale cereale, Malus domestica, Brachypodium distachyon, Hordeum marinum, Aegilops tauschii, Daucus g/ochidiatus, Beta vulgaris, Daucus pusillus, Daucus muricatus, Daucus carota, Eucalyptus grandis, Nicotiana sylvestris, Nicotiana tomentosiformis, Nicotiana tabacum, Solanum lycopersicum, Solanum tuberosum, Coffea canephora, Vitis vinifera, Erythrante guttata, Genlisea aurea, Cucumis sativus, Morus notabilis, Arabidopsis arenosa, Arabidopsis lyrata, Arabidopsis thaliana, Crucihimalaya himalaica, Crucihimalaya wallichii, Cardamine flexuosa, Lepidium virginicum, Capsella bursa pastoris, Olmarabidopsis pumila, Arabis hirsute, Brassica napus, Brassica oeleracia, Brassica rapa, Raphanus sativus, Brassica juncea, Brassica nigra, Eruca vesicaria subsp. sativa, Citrus sinensis, Jatropha curcas, Populus trichocarpa, Medicago truncatula, Cicer yamashitae, Cicer bijugum, Cicer arietinum, Cicer reticulatum, Cicer judaicum, Cajanus cajanifolius, Cajanus scarabaeoides, Phaseo/us vulgaris, Glycine max, Gossypium sp., Astragalus sinicus, Lotus japonicas, Torenia foumieri, Allium cepa, Allium fistulosum, Allium sativum, Helianthus annuus, Helianthus tuberosus and Allium tuberosum, or any variety or subspecies belonging to one of the aforementioned plants.

In another embodiment according to the first aspect of the present invention, the immature inflorescence is a male inflorescence (tassel) of a Zea mays plant, or wherein the immature infloresecene is the inflorescence of a Triticum aestivum plant.

In yet another embodiment of the present invention, the introduction of the genetic construct of interest as defined in step (iv) according to the above aspect is performed at a developmental stage, either during the stamen initiation process or before spikelets are formed on the male inflorescence.

In a second aspect according to the present invention there is provided a method for manufacturing a transgenic plant comprising the following steps: (i) providing an in planta transformed plant or plant material transformed by a method according to the first aspect of the present invention; (ii) cultivating the plant or plant material under suitable conditions until the developmental stage of maturity of the inflorescence is achieved, (iii) allowing the transformed gamete of the pollen or of the ovule generated from the transformed at least one meristematic cell to fuse to a zygote comprising the genetic construct of interest as transgene; and (iv) developing the transgenic plant from the zygote.

In a third aspect according to the present invention there is provided a method for manufacturing a genetically manipulated plant comprising the following steps: (i) providing an in planta transformed plant or plant material transformed by a method according the first aspect of the present invention detailed above; (ii) cultivating the plant or plant material under suitable conditions until the developmental stage of maturity of the inflorescence is achieved; (iii) allowing the genetically manipulated gamete of the pollen or of the ovule generated from the transformed at least one meristematic cell to fuse to a zygote comprising the targeted genetic manipulation; and (iv) developing the genetically manipulated plant from the zygote.

In still another aspect according to the present invention there is provided a plant manufactured by the manufacturing methods of the second or third aspect of the present invention, or plant cells, a plant material, or derivatives or a progeny thereof.

In yet another aspect according to the present invention there is provided a plant, preferably a Zea mays plant, comprising an artificially introduced window in a region, where a meristematic tissue of an immature inflorescence is located, preferably in the axil at the culm of the plant, preferably wherein the plant is a plant in planta transformed by a method according to the first aspect according to the present invention.

There is provided a further aspect according to the present invention which is directed to the use of a plant comprising an artificially introduced window in a region, where a meristematic tissue of an immature inflorescence is located, preferably in the axil at the culm of the plant, preferably wherein the plant is a plant in planta transformed by a method according to the first aspect according to the present invention, or of a plant in planta transformed by a method according to the first aspect according to the present invention, for the manufacture of a transgenic or genetically manipulated plant, plant cell or plant material.

Further aspects and embodiments of the present invention can be derived from the subsequent detailed description, the drawings as well as the attached set of claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 (FIG. 1) shows a schematic diagram of the maize immature inflorescence, the tassel and illustrates details of a spikelet pair. FIG. 1 (A) shows the male inflorescence (tassel) consisting of a central main spike. At the base, long lateral branches branching off the main branch can be found. Short branches called “spikelet pairs” cover both the main spike as well as the lateral branches. FIG. 1 (B) shows a detailed view of a spikelet pair from a normal maize tassel. The spikelet pair is situated at a branch (lateral or main branch). The Sessile Spikelet of the spikelet pair originates from the base where it is attached to whilst the Pedicellate Spikelet is situated on a pedicel. Each spikelet contains two florets, the upper floret and the lower floret enclosed by two glumes, the inner glume and the outer glume. In more detail, each of said florets consists of lemma, palea and two lodicules and three stamens (indicated with a*within each floret).

FIG. 2 A-D (FIG. 2 A-D) shows dissected meristems in maize seedlings. As the meristem is completely covered by leaves in seedlings, it has to be dissected first to be exposed for the further treatment. To this end, the surrounding tissue structures have to be removed to expose the meristem (indicated with an arrow in FIG. 2).

FIG. 3 A-C (FIG. 3 A-C) shows dissected meristems in older maize plants. As the meristem in older plants is completely surrounded by leaves, as for the seedlings shown in FIG. 2, it has to be dissected first without extracting or isolating it to be exposed for a further treatment. To this end the proximal leaves have to be removed to expose the meristem (indicated with an arrow in the respective Figures A to C). Age of the plants in FIG. 3: (A) 8 days, (B) 11 days, (C) 46 days.

FIG. 4 (FIG. 4) shows the tassel development in maize at different days calculated after sowing, i.e. at days 30, 34, 37 and 41, respectively.

FIG. 5 (FIG. 5) exemplary shows the window opening and the tassel exposure for a maize A188 plant. (A) Removal one by one of leaf tissue. (B) Exposed tassel after window opening.

FIG. 6 A-D (FIG. 6 A-D) shows immature tassels detached from the plant and bombarded with a PDS-1000/He gene gun with a genetic construct driving the expression of a red fluorescent protein (RFP). Expression in immature tassels was observed 1 day after bombardment. (A) and (B) whole tassel, (B) fluorescence channel detection; (A) bright field picture of the same structure. White spots and regions in (B) correspond to red fluorescence indicative of expression of the RFP encoded by the genetic construct and transformed into the tassel. (C) and (D) Close up view of the immature spikelets. (D) Again, white spots and regions in (D) correspond to red spots detected corresponding to the expression of the RFP in the immature spikelet.

FIG. 7 A-F (FIG. 7 A-F) shows the results of in planta bombardment with a construct encoding a red fluorescent protein (RFP). (A) Artificial window opened in the axil of a maize plant exposing the immature tassel. (B) and (C): A piece of tassel 1 day after bombardment showing transient expression of the RFP in immature spikelets (see FIG. 7 (C)—white spots corresponding to RFP expression as visualized via fluorescence imaging; (B) bright field picture of the same structure. (D): development of the plant after window cutting and bombardment. (E) and (F): further development in planta yielding a fertile tassel.

FIG. 8 A-H (FIG. 8 A-H) shows an example for stable transformation (red fluorescent protein (RFP) encoding construct) as achieved by the methods of the present invention. (A) and (B): Picture of a spikelet 7 days after bombardment. (A) Red fluorescence channel visualizing RFP expression, here shown as bright white areas. (B) Bright field picture of the same structure as in (A). (C) to (E) Visualization of a cell of a glume of a spikelet 16 days after bombardment. (C) Red fluorescence channel, (D) green fluorescence channel, (D) bright field. The white structure in (C) corresponds to the red fluorescence observed. (F) to (H) shows the a glume transformed with a genetic construct encoding a RFP 28 days after bombardment. (F) Bright field, (G) red fluorescence channel with the bright white area corresponding to the detected red fluorescence; (H) green fluorescence channel.

FIG. 9 (FIG. 9 A-D) shows the localization of a wheat immature inflorescence in two different wheat cultivars. FIG. 9 (A) shows a shoot of a wheat plant 1. The circle on the paper in which the plant is located indicated the location of the meristem at the bottom of the stem, FIG. 9 (B) is an enlarged view of the meristem of plant 1 in exposed form, FIG. 9 (C) shows a shoot of a wheat plant 2. The circle on the paper on which the plant is located indicated the location of the meristem. In this cultivar, the meristem is located higher and big enough so that it can easily be detected with the eye. FIG. 9 (D) is an enlarged view of the meristem of plant 2 in exposed form.

FIG. 10 A and B (FIG. 10 A and B) shows red fluorescent sectors in an immature tassel after Agrobacterium mediated transformation. FIG. 10 A shows a bright field image of the transformed structure and FIG. 10 B shows the same structure when analyzed for the presence of red fluorescence, whereas bright and white areas correspond to the observed red fluorescent spots.

FIG. 11 (FIG. 11) shows an A188 plant in the V7 stage (left), the subsequent window opening to expose the immature tassel (center) and the injection of an Agrobacterium solution into the tassel area (right).

FIG. 12 A and B (FIG. 12 A and B) exemplify different stages of the embryo meristem pipeline disclosed herein. FIG. 12 A shows bombarded immature maize embryos transformed with CRISPR constructs as well as a red fluorescent marker. FIG. 12 B shows the embryos 1 day after bombardment monitored for red fluorescence development (white/bright spots corresponding to areas of fluorescence). The treated embryos germinated and were further positive for fluorescence in the pre-screen. The plants grown from the embryos developed tassels and ears and reached the stage of maturity.

FIG. 13 shows an immature B. vulgaris embryo isolated for the purpose of the present invention.

FIG. 14 A and B (FIG. 14 A and B) shows the preparation of B. vulgaris plantelets to gain access to meristematic areas to be transformed. FIG. 14 A shows a mixture of meristem exposures, whereas FIG. 14 B shows a vertically cut meristematic area exposing the cut area up.

FIG. 15 A and B (FIG. 15 A and B) shows immature embryo targeting in wheat. FIG. 15 A (bright field) shows extracted and bombarded wheat embryos. FIG. 15. B shows the corresponding fluorescence image for detection of red fluorescence (bright and white areas). The embryos germinated and developed viable wheat plantelets after transformation and further cultivation.

FIG. 16 A and B (FIG. 16 A and B) shows the gel analysis of a PCR for verifying the successful integration of DNA inserted into tassel meristematic cells of interest for two different transgenes. FIG. 16 A shows the result for the analysis for transgene 1. From left to right lane 1 shows the molecular marker (1kb plus ladder), lane 2 shows H₂O control, lane 3 shows the positive control, lane 4 shows the wild-type A188 DNA, lanes 5 shows the result for sample 41. FIG. 16 B shows the result for the analysis for transgene 2. From left to right lane 1 shows the molecular marker (50 bp), lane 2 shows H₂O control, lane 3 shows the positive control, lanes 4 shows the result for sample 41, lane 5 shows the wild-type without modification and lane 6 shows a 1 kb molecular marker.

DEFINITIONS

As used herein the term “in planta” or “in planta transformation” means that the actual transformation process, i.e. the introduction of a genetic construct according to the present disclosure, is achieved by transforming a plant, plant cell or plant material which is still connected with the living plant it is derived from. Optionally, said in planta transformation can be conducted with a plant, plant cell, plant tissue or plant material that was specifically prepared or exposed for the transformation process still being in connection with the living plant. Said term is thus used to discriminate the respective methods from in vitro ex planta transformation methods, wherein a plant, plant cell, plant tissue or plant material is first dissected from its natural environment for subsequently being transformed in vitro or ex vivo/ex planta. The terms “transforming” or “transformation” or “transformed” in this context thus comprise any form of biological, chemical or physical introduction of at least one genetic construct or molecule by means of, for example, transformation, transfection, (micro)injection, biolistic bombardment, viral infection and the like.

The term “in vitro culture free” as used according to the present disclosure thus implies that no in vitro culture is necessary for the respective process step. This implies that the respective process does directly take place in planta and not in an explanted or dissected plant cell, tissue, organ or material. The present invention in certain embodiments can comprise in vitro steps, e.g. analytical steps for analyzing the stable integration or the transient introduction of a genetic construct of interest according to the present disclosure. In vitro steps can also occur to cultivate a specific transformed plant material, if desired, depending on the desired plant, plant cell, tissue, organ or material to be obtained. Whenever the term “in vitro culture free” is used, the respective transformation method or the manufacturing process or a product obtainable therefrom, however, can completely proceed in planta without necessitating an in vitro culture step for propagating a plant cell, tissue or material.

A “CRISPR system” as used herein refers to any CRISPR system characterized. Presently, five types (I-V) of CRISPR systems have been described (Barrangou et al., 2007, Science, 315(5819):1709-12.; Brouns et al., 2008, Science, 321(5891):960-4.; Marraffini and Sontheimer, 2008, Science, 322(5909):1843-5; Makarova et al., Nature Rev. Microbiol., 13, 722-736, 2015), wherein each system comprises a cluster of CRISPR-associated (cas or others, e.g. cpf) genes and a corresponding CRISPR-array. These characteristic CRISPR arrays consist of repetitive sequences (direct repeats), into which short fragments of non-repetitive sequences are intercalated (so-called “spacers”), wherein the spacer elements originate from short fragments of foreign gene material (protospacer). The CRISPR-arrays are subsequently transcribed to yield short CRISPR RNAs (crRNAs), wherein the crRNAs direct the Cas proteins or other effector nucleases of a CRISPR system to the respective target nucleic acid to be cut, whereas directing works via Watson-Crick base pairing. The Type I and Type III CRISPR systems use complexes made of cas proteins and crRNAs to mediate recognition and subsequent cleavage of target nucleic acid sequences (Wiedenheft et al., 2011, Nature, 477(7365):486-9). In contrast thereto, Type II CRISPR systems in their natural environment act on target DNA by an orchestrated action of the RNA-guided nuclease Cas9 together with two non-coding RNAs, a crRNA and a trans-activating RNA (tracrRNA) (Garneau et al., 2010; Sapranauskas et al., 2011, Nucleic Acids Res., 39(21):9275-82; Deltcheva et al., 2011, Nature, 471(7340):602-7). A possible Type IV CRISPR system was proposed as well (Makraova et al., Biol. Direct, 6 (38), 2011). In addition, a Type V CRISPR system or CRISPR/Cpf1 system has been described recently (Zetsche et al., “Cpf1 Is a Singel RNA-Guides Endonuclease of a Class 2 CRISPR-Cas System”, Cell, 163, pp. 1-13, October 2015; Makarova et al., Nature Rev. Microbiol., 2015, supra). In contrast to the Cas9 nuclease of a Type II CRISPR system, Cpf1 recognizes T-rich Pam sequences and cuts the target DNA in a way resulting in so-called “sticky-ends”, whereas the native Cas9 protein leaves so-called “blunt-end” cuts. Like Cas9 nucleases also Cpf1 contains a RuvC-like endonuclease domain, however, the second HND endeonuclease domain present in Cas9 is lacking from Cpf1 (Makarova & Koonin, Methods Mol. Biol., 1311, 47-75, 2015).

A CRISPR system for use according to the methods of the present invention is a recombinant CRISPR system using a CRISPR nuclease or a variant, e.g. a variant creating a nickase or a nuclease-deficient nuclease, or a catalytically active fragment of a CRISPR nuclease which acts together with an artificial guide RNA or gRNA.

The terms “guide RNA” and “gRNA” are used interchangeably herein. The gRNA can combine the function of a crRNA and/or a tracrRNA. Depending on the assay of interest at least one gRNA, but also more gRNAs can be used to direct at least one CRISPR nuclease or a variant thereof.

The term “construct”, especially “genetic construct” or “recombinant construct” (used interchangeably herein) as used herein refers to a construct comprising, inter alia, plasmids or plasmid vectors, cosmids, artificial yeast- or bacterial artificial chromosomes (YACs and BACs), phagemides, bacterial phage based vectors, an expression cassette, isolated single-stranded or double-stranded nucleic acid sequences, comprising DNA and RNA sequences, or amino acid sequences, viral vectors, including modified viruses, and a combination or a mixture thereof, for introduction or transformation, transfection or transduction into a target cell or plant, plant cell, tissue, organ or material according to the present disclosure. A recombinant construct according to the present invention can comprise an effector domain, either in the form of a nucleic acid or an amino acid sequence, wherein an effector domain represents a molecule, which can exert an effect in a target cell and includes a transgene, an single-stranded or double-stranded RNA molecule, including a guideRNA, a miRNA or an siRNA, or an amino acid sequences, including, inter alia, an enzyme or a catalytically active fragment thereof, a binding protein, an antibody, a transcription factor, a nuclease, preferably a site specific nuclease, and the like. Furthermore, the recombinant construct can comprise regulatory sequences and/or localization sequences. The recombinant construct can be integrated into a vector, including a plasmid vector, and/or it can be present isolated from a vector structure, for example, in the form of a polypeptide sequence or as a non-vector connected single-stranded or double-stranded nucleic acid. After its introduction, e.g. by transformation, the genetic construct can either persist extrachromosomally, i.e. non integrated into the genome of the target cell, for example in the form of a double-stranded or single-stranded DNA, a double-stranded or single-stranded RNA or as an amino acid sequence. Alternatively, the genetic construct, or parts thereof, according to the present disclosure can be stably integrated into the genome of a target cell, including the nuclear genome or further genetic elements of a target cell, including the genome of plastids like mitochondria or chloroplasts. The term “plasmid vector” as used in this connection refers to a genetic construct originally obtained from a plasmid. A plasmid usually refers to a circular autonomously replicating extrachromosomal element in the form of a double-stranded nucleic acid sequence. In the field of genetic engineering these plasmids are routinely subjected to targeted modifications by inserting, for example, genes encoding a resistance against an antibiotic or an herbicide, a gene encoding a target nucleic acid sequence, a localization sequence, a regulatory sequence, a tag sequence, a marker gene, including an antibiotic marker or a fluorescent marker, and the like. The structural components of the original plasmid, like the origin of replication, are maintained. According to certain embodiments of the present invention, the localization sequence can comprise a nuclear localization sequence, a plastid localization sequence, preferably a mitochondrion localization sequence or a chloroplast localization sequence. Said localization sequences are available to the skilled person in the field of plant biotechnology. A variety of plasmid vectors for use in different target cells of interest is commercially available and the modification thereof is known to the skilled person in the respective field.

A “fertile plant” is a plant that can produce viable male and female gametes and is self-fertile. Such a self-fertile plant can produce a progeny plant without the contribution from any other plant of a gamete and the genetic material contained therein. A plant that is “not self-fertile” thus means a plant that cannot produce male gametes, or female gametes, or both, that are viable or otherwise capable of fertilization. As used herein, a “male sterile plant” is a plant that does not produce male gametes that are viable or otherwise capable of fertilization. As used herein, a “female sterile plant” is a plant that does not produce female gametes that are viable or otherwise capable of fertilization. It is recognized that male-sterile and female-sterile plants can be female-fertile and male-fertile, respectively. It is further recognized that a male fertile (but female sterile) plant can produce viable progeny when crossed with a female fertile plant and that a female fertile (but male sterile) plant can produce viable progeny when crossed with a male fertile plant.

The term “genetically modified” or “genetic manipulation” or “genetic(ally) manipulated” is used in a broad sense herein and means any modification of a nucleic acid sequence or an amino acid sequence, a target cell, tissue, organ or organism, which is accomplished by human intervention, either directly or indirectly, to influence the endogenous genetic material or the transciptome or the proteinome of a target cell, tissue, organ or organism to modify it in a purposive way so that it differs from its state as found without human intervention. The human intervention can either take place in vitro or in vivo/in planta, or also both. Further modifications can be included, for example, one or more point mutation(s), e.g. for targeted protein engineering or for codon optimization, deletion(s), and one or more insertion(s) or deletion(s) of at least one nucleic acid or amino acid molecule (including also homologous recombination), modification of a nucleic acid or an amino acid sequence, or a combination thereof. The terms shall also comprise a nucleic acid molecule or an amino acid molecule or a host cell or an organism, including a plant or a plant material thereof which is/are similar to a comparable sequence, organism or material as occurring in nature, but which have been constructed by at least one step of purposive manipulation.

A “targeted genetic manipulation” as used herein is thus the result of a “genetic manipulation”, which is effected in a targeted way, i.e. at a specific position in a target cell and under the specific suitable circumstances to achieve a desired effect in at least one cell, preferably a plant cell, to be manipulated.

The term “transgenic” as used according to the present disclosure refers to a plant, plant cell, tissue, organ or material which comprises a gene or a genetic construct, comprising a “transgene” that has been transferred into the plant, the plant cell, tissue organ or material by natural means or by means of genetic engineering techniques from another organism. The term “transgene” comprises a nucleic acid sequence, including DNA or RNA, or an amino acid sequence, or a combination or mixture thereof. Therefore, the term “transgene” is not restricted to a sequence commonly identified as “gene”, i.e. a sequence encoding protein. It can also refer, for example, to a non-protein encoding DNA or RNA sequence. Therefore, the term “transgenic” generally implies that the respective nucleic acid or amino acid sequence is not naturally present in the respective target cell, including a plant, plant cell, tissue, organ or material. The terms “transgene” or “transgenic” as used herein thus refer to a nucleic acid sequence or an amino acid sequence that is taken from the genome of one organism, or produced synthetically, and which is then introduced into another organism, in a transient or a stable way, by artificial techniques of molecular biology, genetics and the like.

The term “plant” or “plant cell” as used herein refers to a plant organism, a plant organ, differentiated and undifferentiated plant tissues, plant cells, seeds, and derivatives and progeny thereof. Plant cells include without limitation, for example, cells from seeds, from mature and immature embryos, meristematic tissues, seedlings, callus tissues in different differentiation states, leaves, flowers, roots, shoots, gametophytes, sporophytes, pollen and microspores, protoplasts, macroalgae and microalgae. The different plant cells can either be haploid, diploid or multiploid.

A “plant material” as used herein refers to any material which can be obtained from a plant during any developmental stage. The plant material can be obtained either in planta or from an in vitro culture of the plant or a plant tissue or organ thereof. The term thus comprises plant cells, tissues and organs as well as developed plant structures as well as sub-cellular components like nucleic acids, polypeptides and all chemical plant substances or metabolites which can be found within a plant cell or compartment and/or which can be produced by the plant, or which can be obtained from an extract of any plant cell, tissue or a plant in any developmental stage. The term also comprises a derivative of the plant material, e.g. a protoplast, derived from at least one plant cell comprised by the plant material. The term therefore also comprises meristematic cells or a meristematic tissue of a plant.

The term “transient introduction” as used herein refers to the transient introduction of at least one recombinant construct according to the present disclosure into a plant target structure, for example, a plant cell, wherein the at least one recombinant construct is introduced under suitable reaction conditions so that no integration of the at least one recombinant construct into the endogenous nucleic acid material of a target plant structure, i.e. the plant genome as a whole, occurs, so that the at least one recombinant construct will not be integrated into the endogenous DNA of the target cell. As a consequence, in the case of transient introduction, the introduced genetic construct will not be inherited to a progeny of the plant target structure, for example the plant cell. The at least one recombinant or genetic construct or the products resulting from transcription or translation thereof are only present temporarily, i.e. in a transient way, in constitutive or inducible form, and thus can only be active in the target cell for exerting their effect for a limited time. Therefore, the at least one genetic or recombinant construct introduced via transient introduction will not be heritable to the progeny of a cell. The effect which a recombinant construct introduced in a transient way will cause, can, however, potentially be inherited to the progeny of the target cell.

The term “stable integration” or “stably integrated” as used herein, refers to the stable integration of at least one recombinant or genetic construct according to the present disclosure. The integration can either take place into the nuclear genome or any other genomic extra-nuclear material within a plant compartment of interest, e.g. a mitochondrium. A stably integrated at least one recombinant construct will thus be heritable to the progeny of a thus modified target cell. Depending on the nature of the genetic construct, all or part of the genetic construct will be stably integrated, as the genetic construct may comprise several regions of interest comprising a target region to be stably integrated as well as further regions, inter alia, needed for the transport, delivery, maintenance, and the correct localization of the genetic construct within a plant cell, which regions, however, will not themselves be integrated, but serve as cargo for the region of interest to be stably integrated as it is known to the skilled person. The stable integration of at least one genetic construct according to the present disclosure into at least one meristematic cell or tissue will consequently lead to the inheritance of the thus modified genomic region of the plant target structure to the progeny of the modified cell through all developmental stages of said at least one meristematic cell, which can be favorable for approaches, where a targeted genetic modification in and the yield of the final cell type resulting from the differentiation and development of the at least one meristematic cell is desired. Achieving a stable integration into at least one meristematic cell of the immature inflorescence of a plant can thus lead to the stable inheritance of the introduced genetic feature into the gamete of the pollen or of the ovule developmentally resulting from the at least one meristematic cell of the immature inflorescence.

The term “particle bombardment” as used herein, also named “biolistic transfection” or “microparticle-mediated gene transfer”, refers to a physical delivery method for transferring a coated microparticle or nanoparticle comprising a nucleic acid or a genetic construct of interest into a target cell or tissue. The micro or nanoparticle functions as projectile and is fired on the target structure of interest under high pressure using a suitable device, often called gene-gun. The transformation via particle bombardment uses a microprojectile of metal covered with the gene of interest, which is then shot onto the target cells using an equipment known as “gene gun” (Sandford et al. 1987) at high velocity fast enough (˜1500 km/h) to penetrate the cell wall of a target tissue, but not harsh enough to cause cell death. For protoplasts, which have their cell wall entirely removed, the conditions are different logically. The precipitated nucleic acid or the genetic construct on the at least one microprojectile is released into the cell after bombardment, and integrated into the genome. The acceleration of microprojectiles is accomplished by a high voltage electrical discharge or compressed gas (helium). Concerning the metal particles used it is mandatory that they are non-toxic, non-reactive, and that they have a lower diameter than the target cell. The most commonly used are gold or tungsten. There is plenty of information publicly available from the manufacturers and providers of gene-guns and associated system concerning their general use.

The term “derivative” or “descendant” or “progeny” as used herein in the context of a plant or plant cell or plant material according to the present application relates to the descendants of such a plant or plant cell or plant material which result from natural reproductive propagation including sexual and asexual propagation. It is well known to the person having skill in the art that said propagation can lead to the introduction of mutations into the genome of an organism resulting from natural phenomena which results in a descendant or progeny, which is genomically different to the parental plant or plant cell or plant material, however, still belongs to the same genus/species and possesses the same characteristics as the parental recombinant host cell. Such derivatives or descendants or progeny resulting from natural phenomena during reproduction or regeneration are thus comprised by the term present disclosure.

The term “vector” as used herein refers to a means of transport to deliver a genetic or a recombinant construct according to the present disclosure into a target cell, tissue, organ or plant. A vector thus comprises nucleic acid sequences, optionally comprising sequences like regulatory sequences or localization sequences for delivery, either directly or indirectly, into a target cell of interest or into a plant target structure in the desired cellular compartment of a plant. A vector can also be used to introduce an amino acid sequence into a target cell or target structure. Usually, a vector as used herein can be a plasmid vector. The term “direct introduction” implies that the desired target cell or target structure containing a nucleic acid sequence to be modified according to the present disclosure is directly transformed or transduced or transfected into the specific target cell of interest, where the material delivered with the vector will exert it effect. The term “indirect introduction” implies that the introduction is achieved into a structure, for example, cells of leaves or cells of plant organs or tissues, which do not themselves represent the actual target cell or structure of interest to be transformed, but those structures serve as basis for the systemic spread and transfer of the vector, preferably comprising a genetic construct according to the present disclosure to the actual plant target structure, for example, a meristematic cell or tissue. In case the term “vector” is used in the context of transfecting amino acid sequences into a target cell the term “vector” implies suitable agents for peptide or protein transfection, like for example ionic lipid mixtures. In the context of the introduction of nucleic acid material, the term “vector” cannot only imply plasmid vectors but also suitable carrier materials which can serve as basis for the introduction of nucleic acid or amino acid sequence delivery into a target cell of interest, for example by means of particle bombardment. Said carrier material comprises, inter alia, gold or tungsten particles. Finally, the term “vector” also implies the use of viral vectors for the introduction of at least one genetic construct according to the present disclosure like, for example, modified viruses for example derived from the following virus strains: Maize Streak Virus (MSV), Barley Stripe Mosaic Virus (BSMV), Brome Mosaic virus (BMV, accession numbers: RNA1: X58456; RNA2: X58457; RNA3: X58458), Maize stripe virus (MSpV), Maize rayado fino virus (MYDV), Maize yellow dwarf virus (MYDV), Maize dwarf mosaic virus (MDMV), positive strand RNA viruses of the family Benyviridae, e.g. Beet necrotic yellow vein virus (accesion numbers: RNA1: NC_003514; RNA2: NC_003515; RNA3: NC_003516; RNA4: NC_003517) or of the family Bromoviridae, e.g. viruses of the genus Alfalfa mosaic virus (accesion numbers: RNA1: NC_001495; RNA2: NC_002024; RNA3: NC_002025) or of the genus Bromovirus, e.g. BMV (supra), or of the genus Cucumovirus, e.g. Cucumber mosaic virus (accesion numbers: RNA1: NC_002034; RNA2: NC_002035; RNA3: NC_001440), or of the genus Oleavirus, dsDNA viruses of the family Caulimoviridae, particularly of the family Badnavirus or Caulimovirus, e.g. different Banana streak viruses (e.g. accesion numbers: NC_007002, NC_015507, NC_006955 or NC_003381) or Cauliflower mosaic virus (accesion number: NC_001497), or viruses of the genus Cavemovirus, Petuvirus, Rosadnavirus, Solendovirus, Soymovirus or Tungrovirus, positive strand RNA viruses of the family Closteroviridae, e.g. of the genus Ampelovirus, Crinivirus, e.g. Lettuce infectious yellows virus (accesion numbers: RNA1: NC_003617; RNA2: NC_003618) or Tomato chlorosis virus (accesion numbers: RNA1: NC_007340; RNA2: NC_007341), Closterovirus, e.g. Beet yellows virus (accesion number: NC_001598), or Velarivirus, single stranded DNA (+/−) viruses of the family Geminiviridae, e.g. viruses of the family Becurtovirus, Begomovirus, e.g. Bean golden yellow mosaic virus, Tobacco curly shoot virus, Tobacco mottle leaf curl virus, Tomato chlorotic mottle virus, Tomato dwarf leaf virus, Tomato golden mosaic virus, Tomato leaf curl virus, Tomato mottle virus, oder Tomato yellow spot virus, or Geminiviridae of the genus Curtovirus, e.g. Beet curly top virus, or Geminiviridae of the genus Topocuvirus, Turncurtvirus or Mastrevirus, z. B Maize streak virus (supra), Tobacco yellow dwarf virus, Wheat dwarf virus, positive strand RNA viruses of the family Luteoviridae, e.g. of the genus Luteovirus, e.g. Barley yellow dwarf virus-PAV (accesion number: NC_004750), or of the genus Polerovirus, e.g. Potato leafroll virus (accesion number: NC_001747), single stranded DNA viruses of the family Nanoviridae, comprising the genus Nanovirus or Babuvirus, double stranded RNA viruses of the family Partiviridae, comprising inter alia the families Alphapartitivirus, Betapartitivirus or Deltapartitivirus, viroids of the family Pospiviroidae, positive strand RNA viruses of the family Potyviridae, e.g. comprising the genus Brambyvirus, Bymovirus, Ipomovirus, Macluravirus, Poacevirus, e.g. Triticum mosaic virus (accesion number: NC_012799), or Potyviridae of the genus Potyvirus, e.g. Beet mosaic virus (accesion number: NC_005304), Maize dwarf mosaic virus (accesion number: NC_003377), Potato virus Y (accesion number: NC_001616), or Zea mosaic virus (accesion number: NC_018833), or Potyviridae of the genus Tritimovirus, e.g. Brome streak mosaic virus (accesion number: NC_003501) or Wheat streak mosaic virus (accesion number: NC_001886), single stranded RNA viruses of the family Pseudoviridae, e.g. of the genus Pseudovirus, or Sirevirus, double stranded RNA viruses of the family Reoviridae, e.g. Rice dwarf virus (accesion numbers: RNA1: NC_003773; RNA2: NC_003774; RNA3: NC_003772; RNA4: NC_003761; RNA5: NC_003762; RNA6: NC_003763; RNA7: NC_003760; RNA8: NC_003764; RNA9: NC_003765; RNA10: NC_003766; RNA11: NC_003767; RNA12: NC_003768), positive strand RNA viruses of the family Tombusviridae, e.g. comprising the genus Alphanecrovirus, Aureusvirus, Betanecrovirus, Carmovirus, Dianthovirus, Gallantivirus, Macanavirus, Machlomovirus, Panicovirus, Tombusvirus, Umbravirus oder Zeavirus, e.g. Maize necrotic streak virus (accesion number: NC_007729), or positive strand RNA viruses of the family Virgaviridae, e.g. viruses of the genus Furovirus, Hordeivirus, e.g. Barley stripe mosaic virus (accesion numbers: RNA1: NC_003469; RNA2: NC_003481; RNA3: NC_003478), or of the genus Pecluvirus, Pomovirus, Tobamovirus oder Tobravirus, e.g. Tobacco rattle virus (accesion numbers: RNA1: NC_003805; RNA2: NC_003811), as well as negative strand RNA viruses of the order Mononegavirales, particularly of the family Rhabdoviridae, e.g. Barley yellow striate mosaic virus (accesion number: KM213865) or Lettuce necrotic yellows virus (accesion number/specimen: NC_007642/AJ867584), positive strand RNA viruses of the order Picornavirales, particularly of the family Secoviridae, e.g. of the genus Comovirus, Fabavirus, Nepovirus, Cheravirus, Sadwavirus, Sequivirus, Torradovirus, or Waikavirus, positive strand RNA viruses of the order Tymovirales, particularly of the family Alphaflexiviridae, e.g. viruses of the genus Allexivirus, Lolavirus, Mandarivirus, or Potexvirus, Tymovirales, particularly of the family Betaflexiviridae, e.g. viruses of the genus Capillovirus, Carlavirus, Citrivirus, Foveavirus, Tepovirus, or Vitivirus, positive strand RNA viruses of the order Tymovirales, particularly of the family Tymoviridae, e.g. viruses of the order Maculavirus, Marafivirus, or Tymovirus.

Further suitable vectors can be bacterial vectors, like for example Agrobacterium spp., like for example Agrobacterium tumefaciens. Finally, the term “vector” also implies suitable transport agents for introducing linear nucleic acid sequences (single or double-stranded) into a target cell. The usual production processing and use of vectors as used according to the present disclosure can be accomplished by the person skilled in the art having knowledge of the present disclosure.

The term “target region”, “target site”, “target structure”, “target construct”, “target nucleic acid” or “target cell/tissue/organism” as used herein refers to a target which can be any genomic region within a target cell, but it also refers to extrachromosomal DNA or RNA, including mRNA, of a target cell or organism, or any structural element on or within a target cell, where a modification or manipulation is desired. The term “target region”, “target site”, “target structure”, “target construct”, “target nuclec acid” or “target cell/tissue/organism” is thus not restricted to genomic regions encoding a gene, i.e. a region encoding the information for being transcribed to an mRNA.

“Complementary” or “complementarity” as used herein describes the relationship of two DNA or RNA nucleic acid regions. Defined by the nucleobases of the DNA or RNA two nucleic acid regions can hybridize to each other in accordance with the lock-and-key model. To this end the principles of Watson-Crick base pairing have the basis adenine and thymine/uracil as well as guanine and cytosine, respectively, as complementary basis apply. Furthermore, also non-Watson-Crick pairing, like reverse-Watson-Crick, Hoogsteen, reverse-Hoogsteen and Wobble pairing are comprised by the term “complementary” as used herein as long as the respective base pairs can build hydrogen bonding to each other, i.e. two different nucleic acid strands can hybridize to each other based on said complementarity.

The term “regulatory sequence” as used herein refers to a nucleic acid or amino acid sequence, which can direct the transcription and/or translation and/or modification of a nucleic acid sequence of interest. Regulatory sequences can comprise sequences acting in cis or acting in trans. Exemplary regulatory sequences comprise promoters, enhancers, terminators, operators, transcription factors, transcription factor binding sites, introns and the like.

A “promoter” refers to a DNA sequence capable of controlling expression of a coding sequence, i.e. a gene or part thereof, or of a functional RNA, i.e. a RNA which is active without being translated, for example, a miRNA, a siRNA, a IncRNA, an inverted repeat RNA or a hairpin forming RNA. The promoter sequence consists of proximal and distal elements in relation to the regulated sequence, the latter being often referred to as enhancers. Promoters can have a broad spectrum of activity, but they can also have tissue or developmental stage specific activity. For example, they can be active in cells of roots, seeds and meristematic cells, etc. A promoter can be active in a constitutive way, or it can be inducible. The induction can be stimulated by a variety of environmental conditions and stimuli. There exist strong promoters which can enable a high transcription of the regulated sequence, and weak promoters. Often promoters are highly regulated. A promoter of the present disclosure may include an endogenous promoter natively present in a cell, or an artificial, a synthetic (chimeric) or a transgenic promoter, either from another species, or an artificial or chimeric promoter, i.e. a promoter that does not naturally occur in nature in this composition and is composed of different promoter elements.

The term “terminator”, as used herein, refers to DNA sequences located downstream, i.e. in 3′ direction, of a coding sequence and can include a polyadenylation signal and other sequences, i.e. further sequences encoding regulatory signals that are capable of affecting mRNA processing and/or gene expression. The polyadenylation signal is usually characterized in that it adds poly-A-nucleotides at the 3′ end of an mRNA precursor.

The term “nucleic acid (molecule)” or “nucleic acid construct”or “nucleic acid sequence” as used herein refers to natural and synthetic deoxyribonucleic acids (DNA) or ribonucleic acid (RNA) sequences, which can optionally comprise synthetic nucleic acid analoga. A nucleic acid according to the present disclosure can optionally be codon optimized. Codon optimization implies that the codon usage of a DNA or RNA is adapted to that of a cell or organism of interest to improve the transcription rate of said recombinant nucleic acid in the cell or organism of interest. The skilled person is well aware of the fact that a target nucleic acid can be modified at one position due to the codon degeneracy, whereas this modification will still lead to the same amino acid sequence at that position after translation, which is achieved by codon optimization to take into consideration the species-specific codon usage of a target cell or organism. Nucleic acid sequences according to the present application can carry specific codon optimization for the following non limiting list of organisms: Hordeum vulgare, Hordeum bulbusom, Sorghum bicolor, Saccharum officinarium, Zea mays, Setaria italica, Oryza minuta, Oryza sativa, Oryza australiensis, Oryza alta, Triticum aestivum, Triticale, Secale cereale, Malus domestica, Brachypodium distachyon, Hordeum marinum, Aegilops tauschii, Daucus glochidiatus, Beta vulgaris, Daucus pusillus, Daucus muricatus, Daucus carota, Eucalyptus grandis, Nicotiana sylvestris, Nicotiana tomentosiformis, Nicotiana tabacum, Solanum lycopersicum, Solanum tuberosum, Coffea canephora, Vitis vinifera, Erythrante guttata, Genlisea aurea, Cucumis sativus, Morus notabilis, Arabidopsis arenosa, Arabidopsis lyrata, Arabidopsis thaliana, Crucihimalaya himalaica, Crucihimalaya wallichii, Cardamine flexuosa, Lepidium virginicum, Capsella bursa pastoris, Olmarabidopsis pumila, Arabis hirsute, Brassica napus, Brassica oeleracia, Brassica rapa, Raphanus sativus, Brassica juncea, Brassica nigra, Eruca vesicaria subsp. sativa, Citrus sinensis, Jatropha curcas, Populus trichocarpa, Medicago truncatula, Cicer yamashitae, Cicer bijugum, Cicer arietinum, Cicer reticulatum, Cicer judaicum, Cajanus cajanifolius, Cajanus scarabaeoides, Phaseolus vulgaris, Glycine max, Gossypium sp., Astragalus sinicus, Lotus japonicas, Torenia foumieri, Allium cepa, Allium fistulosum, Allium sativum, Helianthus annuus, Helianthus tuberosus and Allium tuberosum, or any variety or subspecies belonging to one of the aforementioned plants.

The terms “protein”, “polypeptide” and “enzyme” are used interchangeably herein. The term “amino acid” or “amino acid sequence” or “amino acid molecule” comprises any natural or chemically synthesized protein, peptide, polypeptide and enzyme or a modified protein, peptide, polypeptide and enzyme, wherein the term “modified” comprises any chemical or enzymatic modification of the protein, peptide, polypeptide and enzyme.

The term “homologous recombination” as used herein refers to a process, as it naturally occurs in all organisms. This requires homologous double stranded DNA segments. “Homologue” therefore means that there is a great similarity between two nucleotide sequences. In naturally occurring double-strand breaks, the damage can thus be repaired by homologous recombination and the information on the undamaged chromatid in the genome of an organism can be used as a template. In accordance with the present disclosure, a directed and precise double-strand break or a double nick during the course of genome editing method performed in planta can be inserted into a nucleic acid target region of interest and homologous recombination can then be used to repair the resulting break. To this end a specifically designed DNA repair template or HDR template can be transformed into at least one meristematic cell of a meristematic tissue of a plant of interest. Different organisms differ in terms of the ratio of homologous and non-homologous recombination (NHEJ) as it naturally occurs. In general, the length of the homology region influences the frequency of homologous recombination events, the longer the region of homology, the greater the frequency. The length of the region of homology required to achieve homologous recombination is species dependent. In some cases it may be necessary to use at least five kilobases (kb) of homology, but homologous recombination has also been observed in a region of homology of only about 25 base pairs (bp).

Homology-directed repair (HDR) denotes a cellular mechanism to repair double as well as single-stranded DNA breaks. Thus, HDR includes elements of homologous recombination as well as the so-called single-strand annealing (SSA) (Dear Michael et al, Annu. Rev. Biochem.79. 181-211, 2010). The most common form of HDR in a cell is the homologous recombination, this type of repair also requires the highest sequence homology between donor and acceptor DNA. Other forms of HDR include single strand annealing (SSA). SSA is not conservative and occurs naturally between direct repeats of >30 bp, resulting in deletions. HDR at nicks, i.e. at single-strand breaks, occurs via a mechanism other than HDR at double strand breaks (Davis and Maizels PNAS, 2014 E924-32). According to the present disclosure, both double-stranded and single-stranded break inducing endonucleases, e.g. CRISPR nucleases or variants or catalytically active fragments thereof modified to act as nickase, are proposed. The term HDR or homologous recombination, therefore, relates to the repair of a specifically introduced single-strand as well as double strand break(s) using a suitable repair mechanism.

Herbicide resistance or herbicide tolerance as used herein refers to the ability of a plant or a plant cell to develop resistance or tolerance to a certain herbicide or pesticide. This property is commonly imparted by introducing at least one protein, or a functional RNA, which was either introduced into a plant cell, or which protein or RNA can be obtained by targeted modification of an endogenous gene or non-coding sequence.

The term genome as used herein refers to the sum of the genetic material of a cell, comprising coding and non-coding genes being present in the cell of an organism or a virus or an organelle of an organism. The term thus comprises the nuclear (if present) genome as well as extra-nuclear genomic material.

DETAILED DESCRIPTION

According to a first aspect of the present invention, there is provided a method for the in planta transformation of a plant or plant material with a genetic construct of interest comprising the following steps: (i) providing a plant comprising at least one meristematic cell of a meristematic tissue of an immature inflorescence, wherein the meristematic cell is able to differentiate into a gamete of a pollen or of an ovule; (ii) exposing the at least one meristematic cell of the meristematic tissue of the immature inflorescence; (iii) providing a genetic construct of interest; and (iv) transforming the at least one meristematic cell of the meristematic tissue of the immature inflorescence by introducing the genetic construct of interest under suitable conditions to allow the functional integration of the genetic construct of interest into the at least one meristematic cell of the meristematic tissue of the immature inflorescence.

The methods and uses as provided by the present invention and the plant products obtainable therefrom offers an advanced alternative to the current protocols of transforming inter alia the immature inflorescences, e.g. of corn, in planta, producing, for example, transgenic pollen that can be used for pollination, obtaining directly transgenic T1 seeds. Therefore, the methods as provided by the state of the art necessarily have to proceed through in vitro culture, irrespective of whether a stable or a transient modification of the plant is intended. The process according to the present invention is thus fast, genotype independent and in vitro culture free, if desired. Consequently, a transformed plant or plant material can be directly obtained from the plant being modified without cumbersome crossings and cultivation and screening of the F1 progeny. The methods as disclosed herein can be adapted to other flowers or inflorescences of other crops based on the disclosure provided herein.

As illustrated in FIG. 1, the immature inflorescence to be targeted can be the male inflorescence of corn, the tassel. The basic structure of the tassel inflorescence is the spikelet. Two types of spikelets, sessile and pedicellate spikelets, develop in the different branches of the tassel. Each spikelet covers with an outer and inner glume two individual flowers that consist of other two covers, the lemma and the palea that are protecting three stamens, the filament and the anther that will produce pollen, i.e. around 2-25 million pollen grains per tassel. The transformation methods according to the present invention should be done preferably during the stamen initiation process, introducing the foreign DNA in the L2 layer of cells of the stamen which is advantageous as those cells will end up producing transgenic pollen.

“Meristematic cells” as referred to according to the present disclosure belong to a tissue type within a plant which is also referred to as meristem or cambium or formative tissue. Like stem cells in animal organisms, meristematic cells of plants representing undifferentiated cells have the intrinsic capability to develop and differentiate into specialized cell types, depending on genetic predisposition and further environmental and developmental factors. In plant organisms, meristems are not only present during the embryo development, but they can be found during the whole life cycle of a plant so that a targeted genetic modification of meristematic cells or tissues according to the present disclosure is not restricted to plant embryos or seedlings, but it can rather also be conducted in larger seedlings and more matured plants, for example when targeting meristems which build the basis for the reproductive plant organs, for example the tassel or corn cob in maize.

According to one embodiment according to the various aspects according to the present disclosure the at least one meristematic cell is a mature or immature plant cell of a plant embryo or seedling or of a plant comprising at least one meristematic cell or meristematic tissue.

According to one specific embodiment according to the various aspects of the present disclosure, at least one a meristematic cell of a meristematic tissue of an immature inflorescence, wherein the meristematic cell is able to differentiate into a gamete of a pollen or of an ovule. As it is known to the skilled person in the field of plant sciences or plant biotechnology, the inflorescence of a seed plant is part of the shoot axis and involved in flower formation. Furthermore, it is known that there are unisexual (either male or female) and bisexual flower types. Consequently, depending on the flower type said plants can develop pollen or an ovule even in the same or in different flowers. The immature inflorescence thus represents the developmental precursor of the inflorescence comprising developing meristematic cells. Currently, it is common in the literature to perform plant regeneration starting from immature inflorescences ex vivo/ex planta by establishing in vitro callus cultures thereof (see e.g. Praveena and Girl, Physiol. Mol. Biol. Plants, 2012).

The methods according to the present invention further provide a significant improvement to current strategies for using engineered site specific nucleases for targeted and precise genome engineering in different plants of economic interest.

In one embodiment, effector nucleases, e.g. homing endonucleases, meganucleases, zinc finger nucleases, transcription activator-like effector nucleases (TALENs) or CRISPR nucleases, e.g. Cas nucleases or Cpf1 nucleases, or variants or catalytically active fragments thereof, alone or in combination as fusions on at least one genetic construct or introduced via protein transfection can be inserted in planta into at least one meristematic cell of interest together with further regulatory sequences and/or effector molecules. Effector molecules can be composed of DNA, RNA or synthetic molecules, or a combination thereof and can associate, e.g. with a CRISPR nuclease and/or a gRNA. Effector molecules can be DNA-modifying or binding enzymes, or the sequences coding therefore, or sequences mediating epigenetic modifications.

In one embodiment according to any aspect of the present invention, the at least one CRISPR polypeptide, or the nucleotide sequence encoding the same, is independently selected from the group consisting of a Cas polypeptide of Streptococcus spp., including Streptococcus pyogenes, Streptococcus thermophiles, Staphylococcus aureus, or Neisseria spp., including Neisseria meningitides, Corynebacterium, Sutterella, Legionella, Treponema, Filifactor, Eubacterium, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, Sphaerochaeta, Azospirillum, Gluconacetobacter, Roseburia, Parvibaculum, Nitratifractor, Mycoplasma and Campylobacter, including Campylobacter jejuni, or the CRISPR polypeptide, or the nucleotide sequence encoding the same, is selected from a Cpf1 polypeptide derived from an archaea or a bacterium, including a Cpf1 polypeptide of Acidaminococcus spp., including Acidaminococcus sp. BV3L6, Lachnospiraceae spp., including Lachnospiraceae bacterium ND2006, Francisella spp., including Francisella novicida U112, Eubacterium eligens, Prevotella spp., or Porphyromonas spp., or variants and/or functional fragments and/or combinations thereof, including nickases, or a CRISPR polypeptide lacking endonucleolytic activity, or a CRISPR polypeptide fusion protein, wherein one CRISPR polylpeptide moiety can be fused to another CRISPR polypeptide moiety or to any DNA, RNA or polypeptide effector domain.

Furthermore, according to the present disclosure, at least one DNA repair template can be transformed in planta into at least one meristematic cell of interest together with a genome editing tool of interest. The use of such a repair template is of special interest in case an endonuclease or at least one nickase is used to introduce a specific doublestrand break or several nicks into a genomic target site of interest. By providing a suitable repair template assisting in homology directed repair, and the introduction of a modification of interest, the genome editing event can be controlled in an even more precise manner. A DNA double-stranded break in vivo is usually repaired by natural mechanisms in the plant cells: either by “non-homologous end joining” (NHEJ) or by homologous recombination (HR; also known as “homology-directed repair” (HDR)). Furthermore, in plants, so-called alternative end joining (AEJ) pathways have been described (Charbonnel C, Allain E, Gallego M E, White C I (2011) Kinetic analysis of DNA double-strand break repair pathways in Arabidopsis. DNA Repair (Amst) 10: 611-619). As NHEJ is highly error prone, there is a great need in providing repair mechanisms allowing more precision.

In the context of the present disclosure, in one embodiment a repair template for HDR is disclosed which can optionally be introduced into the target cell together with or at a separate time to an endonclease, e.g. a CRISPR construct, and/or a further at least one effector domain, in order to induce a specific HDR mechanism and thus introduce specific sequence changes at the site of a double-stranded break. In this regard, both specific edits, knock-ins, gene exchanges, or specific repair of the DNA lesion to prevent an unwanted mutation at the site of the DNA break may be carried out. A knock-in can mean the specific insertion of at least one nucleotide, at least 2 nucleotides, at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 15 nucleotides, at least 20 nucleotides, at least 50 nucleotides, at least 100 nucleotides, at least 200 nucleotides, at least 500 nucleotides or at least 1000 nucleotides into the target nucleic acid in the plant cell. An edit may mean the specific exchange of at least one nucleotide for another nucleotide. Further, an edit may also be obtained by two, three, four or more exchanges or a combination of insertions and exchanges. “Insertion(s)” means the specific insertion of at least one nucleotide, at least 2 nucleotides, at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 15 nucleotides, at least 20 nucleotides, at least 50 nucleotides, at least 100 nucleotides, at least 200 nucleotides, at least 500 nucleotides, at least 1000 nucleotides, at least 2000 nucleotides, at least 5000 nucleotides, at least 10000 nucleotides or at least 15000 nucleotides into the target nucleic acid of the plant cell. A nucleic acid sequence as an insertion may be all or part of a sequence of a transcription factor binding site, a regulatory sequence, a polypeptide-coding sequence, an intron sequence, a sequence a non-coding RNA (for example IncRNA), an expression cassette, another noncoding sequence with various uses, such as for introduction of a breeding marker at a desired location, or a RNAi construct. Furthermore, a knock-in may also be brought about by a deletion of sequence sections which perturb the functionality of a gene (for example the deletion of transposon insertions). The repair template may be introduced into the target plant structure of interest as a single-stranded or as a double-stranded nucleic acid or a combination thereof. The method according to this first aspect thus allows the functional integration of a genetic construct of interest into the at least one meristematic cell of the meristematic tissue of the immature inflorescence directly in planta and, in one embodiment, allows the further in vivo in planta cultivation of a transformed cell or tissue.

The term “functional integration” or “functionally integrated” as used herein refers to the integration of a genetic construct of interest into at least one meristematic cell of a meristematic tissue which allows the transcription and/or translation and/or the catalytic activity and/or binding activity, including the binding of a nucleic acid molecule to another nucleic acid molecule, including DNA or RNA, or the binding of a protein to a target structure within the at least one meristematic cell. Where pertinent, the functional integration takes place in a certain cellular compartment of the at least one meristematic cell, including the nucleus, the cytosol, the mitochondrium, the chloroplast, the vacuole, the membrane, the cell wall and the like. Consequently, the term “functional integration”—in contrast to the term “stable integration” detailed above—implies that the genetic construct of interest is introduced into the at least one meristematic cell by any means of transformation, transfection or transduction by biological means, including Agrobacterium transformation, or physical means, including particle bombardment, as well as the subsequent step, wherein the genetic construct exerts its effect within or onto the at least one meristematic cell in which it was introduced. Depending on the nature of the genetic construct to be introduced said effect naturally can vary and including, alone or in combination, inter alia, the transcription of a DNA encoded by the genetic construct to a ribonucleic acid, the translation of an RNA to an amino acid sequence, the activity of an RNA molecule within a cell, comprising the activity of a guide RNA, or an miRNA or an siRNA for use in RNA interference, and/or a binding activity, including the binding of a nucleic acid molecule to another nucleic acid molecule, including DNA or RNA, or the binding of a protein to a target structure within the at least one meristematic cell, or including the integration of a sequence delivered via a vector or a genetic construct, either transiently or in a stable way. Said effect can also comprise the catalytic activity of an amino acid sequence representing an enzyme or a catalytically active portion thereof within the at least one meristematic cell and the like. Said effect achieved after functional integration of the genetic construct according to the present disclosure can depend on the presence of regulatory sequences or localization sequences which are comprised by the genetic construct of interest as it is known to the person skilled in the art. As the present invention comprises both embodiments directed to the stable integration of a genetic construct of interest as well as the transient introduction of a genetic construct of interest into at least one meristematic cell, the effect achieved by the functional integration of the genetic construct can further vary depending on the mode of introduction being either stable or transient. Stable integration, as detailed above, relies on the integration of the genetic construct into the genome of the at least one meristematic cell, including the nuclear genome as well as the genome of other plant compartments, whereas the transient introduction implies that the functional integration means the introduction of the genetic construct of interest into the at least one meristematic cell followed by its transcription and/or translation and/or catalytic activity and/or binding activity in the cell without the need of a stable integration into the genome of the meristematic cell.

If a CRISPR system is used as part of a genetic construct according to the present invention, the nuclease component, or a variant or a catalytically active fragment thereof, and the gRNA component, and optionally further components like effector domains or an artificial repair template can be introduced into a target cell of interest in a stable way, or in a transient way. Furthermore, the different components can be introduced on the same or on different genetic construct. The different CRISPR components can thus be transformed in planta either simultaneously or subsequently. For example, the CRISPR nuclease, e.g. a Cas nuclease or a Cpf1 nuclease or a variant or a catalytically active fragment thereof, can be incorporated into at least one cell of a meistematic tissue in a stable way to generate a first plant. The at least one cell of a meristematic tissue of said first plant, in the next generation, can then be transformed in planta, with the further CRISPR component comprising at least one gRNA in a transient way. In another embodiment, the CRISPR components are transformed in planta into the same at least one cell of a meristematic tissue, which allows the rapid and precise generation of a genome editing event of interest. In one embodiment, the components can be provided on/as the same genetic construct, or the components can be provided on/as different genetic constructs, either simultaneously or subsequently. As the CRISPR nuclease component as protein will be more stable than the gRNA component, it could be desirable to transform at least one cell of a meistematic tissue in plant in a way that first a steady level of a CRISPR nuclease is achieved, comprising necessary localization signals, e.g. nuclear or plastidic localization signals, before a gRNA is provided, either in the form of RNA or as a DNA genetic construct transcribable into active RNA.

In certain embodiments, the transformation of at least one meristematic cell of a meristematic tissue of an inflorescence according to the methods of the present invention can comprise the introduction of an additional effector domain, including an effector domain for an epigenetic modulation, or introduction of a DNA repair template. DNA repair templates are highly suitable for precision genome editing when used in combination with a site specific endonuclease or nickase, as the provision of the repair template can assist in providing a targeted homology directe repair event. The term “targeted homology directed repair” according to the present disclosure comprises any type of alterations that can be introduced by the repair template sequence according to the present application, which can independently comprise sequence insertions, edits of at least one sequence position, deletions or rearrangements, the preferable strategy for genome editing approaches in higher eukaryotes presently being insertions or edits, as these strategies allow the targeted knock-in or knock-out of a sequence of interest within a DNA target sequence, or a site-specific modification of at least one sequence.

In one embodiment according to this aspect the nucleic acid target sequence, where the functional integration of the genetic construct of interest takes place is the nuclear genomic DNA. In another embodiment according to this aspect the nucleic acid target sequence into which the functional integration of the genetic construct of interest is mediated is a mitochondrial or a plastid DNA, wherein the genetic construct comprises a localization sequence wherein this sequence mediates the localization of the genetic construct in the respective plant compartment of interest, for example, a mitochondrium or a chloroplast. In yet another embodiment, the functional integration refers to the delivery of a genetic construct of interest into at least one meristematic cell, for example into the cytosol or another plant compartment, without that a stable integration takes place.

In one embodiment according to the above and according to the further aspects of the present invention, the transformation is performed in vitro culture free.

In another embodiment, transformation in planta, transformed plant cell, tissue, organ or material is explanted and further cultivated in vitro.

As detailed above, the methods according to the present invention provide the advantage that both the transformation and the further development of a transformed at least one meristematic cell can take place in planta obviating the need for cumbersome in vitro cultivation steps for the regeneration of a plant or plant material therefrom. In certain embodiments, it might, however, be suitable to explant or dissect a plant cell, tissue, organ or material for further cultivation, screening or testing depending on the specific needs. Several methods for the in vitro cultivation of a plant cell, tissue, organ or material are available to the skilled person.

In another embodiment, the functional integration of the genetic construct of interest into the at least one meristematic cell of the meristematic tissue of the immature inflorescence of step (iv) of the above aspect is performed as (a) a stable integration so that the integrated genetic construct of interest, or a part thereof, is heritable to a progeny, preferably whereby a transformed gamete of the pollen or of the ovule is generated from the at least one transformed meristematic cell and the transformed gamete of the pollen or of the ovule is fused to a zygote from which the progeny comprising the genetic construct of interest is developed; as transgene, or (b) a transient introduction allowing a targeted genetic manipulation of the at least one meristematic cell of the meristematic tissue of the immature inflorescence through the genetic construct of interest or products thereof, wherein the targeted genetic manipulation but not the genetic construct of interest, or a part thereof, is heritable to a progeny, preferably whereby a genetic manipulated gamete of the pollen or of the ovule is generated from the transformed at least one meristematic cell and the genetic manipulated gamete of the pollen or of the ovule is fused to a zygote from which the progeny comprising the targeted genetic manipulation is developed.

The methods according to the present invention are highly suitable to achieve efficient and controlled trait development in a plant of interest, e.g. for introducing or modifying marker genes or for confering certain desired traits to a plant in a short period of time.

Particularly with regard to the targeted development of positive traits in plants, comprising resistance, especially against pests and environmental influences, such as cold, drought, salinity, increased yield, or herbicide resistances, there is a need to provide a reliable method for the selective activation and deactivation or modification of genomic material, as well as the silencing of RNA inside a plant cell of great economic interest within a short period of time without in vitro cultivation or intensive breeding steps.

In a further embodiment of the first aspect of the present invention, there is thus provided an in planta transformation method for producing a plant, a plant material or plant cell, wherein at least one target plant structure comprising at least one meristematic cell, is transformed with at least one gRNA, at least one CRISPR nuclease or catalytically active fragment thereof and/or an effector domain and at least one DNA repair template, wherein a targeted modification at least one nucleic acid target region of interest is obtained, either by modifying an endogenous or by inserting a heterologous, i.e. non-endogenous sequence, wherein the targeted modification concerns, or wherein the heterologous sequence comprises a gene that is selected from a reporter gene, a selectable marker, a resistance gene mediating resistance towards a disease, a herbicide resistance-mediating gene, a gene mediating resistance to insects or nematodes, a gene that is involved in carbohydrate metabolism, a gene which is involved in fatty acid metabolism, a gene that is involved in amino acid metabolism, a gene which is involved in development of the plant, a gene which is involved in the regulation of plant growth, a gene which is involved in yield improvement of a plant material of interest, a gene that is involved in mediating resistance to drought, a gene that participates in to convey cold resistance, a gene involved in imparting heat resistance, a gene that is involved in conveying resistance to a salt or salts or a certain salt concentration, or a gene encoding a functional RNA, wherein the functional RNA selected from the group consisting of a miRNA, a siRNA, or other RNA that can form a inverted repeat structure, for example a ddRNAi construct encoding both, a sense and an anti-sense strand and a connecting hairpin loop.

In one embodiment, new molecular markers can be developed. In relevant crop plants, there is the potential to increase efficiency of targeted plant breeding through marker-assisted selection (MAS). Genetic marker alleles, or alternatively the above-described quantitative trait loci (QTL) alleles are used for this purpose in order to identify plants or plant material, or a plant cell, which contain a desired genotype at one or more locus/loci. It is assumed that they can inherit the desired genotype together with a desired phenotype to the offspring. Genetic markers alleles (or QTL alleles) can also be used to identify plants having a desired genotype at a locus, or at multiple unlinked or linked loci, e.g. a haplotype, of which one assumes that they can pass on to their offspring the desired genotype together with a desired phenotype. For the purpose of marker-assisted selection, the term marker as used herein, therefore, refers to both, markers and QTL loci. Once it has been determined that a desired phenotype and a polymorphic chromosomal locus, for example, a marker locus or a QTL, segregate together, it is possible to use these polymorphic loci, to select for alleles corresponding to a desired phenotype. This procedure is called marker-assisted selection (MAS). For this purpose, a nucleic acid sequence corresponding to the marker nucleic acid in a biological sample of interest of a plant to be analyzed is detected. This detection can be in the form of hybridization of a nucleic acid probe to a label, e.g. using allele-specific hybridization, Southern blot analysis, Northern blot analysis in situ hybridization, hybridization of primers followed by PCR amplification of a region of the marker, or the like, or by any any combination thereof. A variety of methods for the detection of markers is known to the skilled person. After the presence or absence of a specific marker in the biological sample of interest, comprising at least one plant cell, preferably a meristematic cell, was confirmed the corresponding plant is selected and can be used for selective breeding to obtain progeny. Similarly, the methods according to the present invention can be used to analyze a meristematic cell specifically modified in planta for the presence or absence of a specific marker. From this meristematic cell either male or female gametes or germ cells can be obtained. Especially the pollen of a plant thus altered in planta may then be directly used for the subsequent selective breeding.

As in classical plant breeding there exists a need to implement features of interest in a target plant, which encode a high yield and/or other desirable characteristics to develop improved plant varieties. Thus, there is a great need for marker-assisted selection, as this approach simplifies complicated and expensive testing of a large number of samples.

According to the methods of the present disclosure specific phenotypic markers can be introduced in a plant target structure of interest in planta. A “phenotypic marker”, as used herein, refers to a selectable marker faciliating the verification of a plant cell or target structure of interest. Phenotypic markers generally comprise either positive or negative selectable markers, for example, optical markers or (antibiotic) resistance genes. Any marker which is useful for a plant target structure of interest, particularly a meristematic cell, may be used. Selectable markers typically include DNA segments that allow a cell, or a molecule within a cell labeled by a “tag”, optionally under certain conditions, to be identified. Such markers can be encode an activity, which is selected from, but not limited to, production of RNA, peptide, or protein, or the markers may provide a binding site for RNA, peptides, proteins, inorganic and organic compounds or compositions and the like. Exemplary selectable markers include, but are not limited to, DNA segments consisting of restriction enzyme sites, DNA segments that comprise a fluorescent probe, DNA segments encoding products that mediate resistance against otherwise toxic compounds including antibiotics, such as spectinomycin, ampicillin, kanamycin, tetracycline, BASTA, neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), DNA segments that encode products which would be missing in a target plant cell of interest under natural circumstances, such as tRNA genes, auxotrophic markers, and the like, DNA segments that encode products which are readily identifiable, particularly visually perceptible marker, e.g. phenotypic markers such as β-galactosidase, GUS, fluorescent proteins, such as green fluorescent protein (GFP) and other fluorescent proteins, such as blue (CFP), yellow (YFP), or red (RFP) fluorescent proteins and surface proteins. In particular, fluorescent proteins are of interest showing a high fluorescence intensity, since these proteins can also be identified in the deeper layers of tissue, which can be the case for the meristematic in planta targeting approaches of the present invention. Further markers can include new primer sites for PCR, or the incorporation of DNA sequences that can not be modified by restriction endonuclease or other DNA modifying enzymes or effector domains of the present disclosure, DNA sequences coding for specific modifications, such as epigenetic modifications, for example, methylation, or DNA sequences which carry a PAM motif, which can be recognized by an appropriate CRISPR system according to the present disclosure, as well as DNA sequences, in which a PAM motif being present in an endogenous plant genomic sequence, is missing.

In one embodiment according to the methods of the present invention, a gRNA/CRISPR nuclease system is used for introducing one or more polynucleotides of interest or one or more traits of interest into one or more target sites comprising the transformation of at least one meristematic cell in planta by providing one or more gRNAs, one ore more CRISPR nucleases or mutated or truncated variants thereof, and optionally one or more donor DNAs, preferably in the form of at least one DNA repair template to a plant cell within a plant of interest. A fertile plant can be produced from that plant cell that comprises an alteration at said one or more target sites, wherein the alteration is selected from the group consisting of (i) replacement of at least one nucleotide, (ii) a deletion of at least one nucleotide, (iii) an insertion of at least one nucleotide, and (iv) any combination of (i)-(iii). Plants comprising these altered target sites can be crossed with plants comprising at least one gene or trait of interest in the same complex trait locus, thereby further stacking traits in said complex trait locus. In one embodiment, the method comprises a method for producing in a plant a complex trait locus comprising at least two altered target sequences in a genomic region of interest, said method comprising: (a) selecting a genomic region in a plant, wherein the genomic region comprises a first target sequence and a second target sequence; (b) contacting at least one meristematic plant cell in planta with at least a first gRNA, a second polynucleotide, and optionally at least one donor DNA or DNA repair template, and a CRISPR nhuclease, wherein the first and second gRNA and the CRISPR nuclease can form a complex that enables the at least one CRISPR nuclease to introduce a double strand break, directly or by introducing two nicks, in at least a first and a second target sequence; (c) identifying a cell from (b) comprising a first alteration at the first target sequence and a second alteration at the second target sequence; and (d) recovering a first fertile plant from the cell of (c) said fertile plant comprising the first alteration and the second alteration, wherein the first alteration and the second alteration are physically linked.

In yet another embodiment according to to the methods of the present invention there is provided a method for producing a male sterile or male fertile plant, the method comprising: (a) obtaining a plant comprising at least one CRISPR nuclease capable of introducing a double strand break, or capable of introducing at least two nicks, at a genomic target site located in a male fertility gene locus in the plant genome of a meristematic cell targeted in planta; (b) providing at least one gRNA that is capable of forming a complex with the at least one CRISPR nuclease of (a), (c) further cultivating the plant until reproductive organs can be harvested (b); d) evaluating the progeny of (c) for an alteration in the target site; and (e) selecting a progeny plant that is male sterile or male fertile. Male fertility genes can be selected from, but are not limited to MS26, MS45, MSCA1 genes. This approach provides the advantage that the genome editing can be directly performed in one plant without the need of crossing a plant comprising a CRISPR nuclease with a plant comprising a gRNA. In another embodiment, the above method can also be performed by a) obtaining a first plant comprising at least one CRISPR nuclease capable of introducing a double strand break, or at least two nicks, at a genomic target site located in a male fertility gene locus in the plant genome of a meristematic cell targeted in planta; (b) providing a second plant comprising at least one gRNA that is capable of forming a complex with the at least one CRISPR nulcease (a), (c) crossing the first plant of (a) with the second plant of (b) and finally (d) evaluating the progeny for the presence of a genome editing event.

Therefore, in some embodiments according to the methods of the present invention the genomic target site within at least one meristematic cell to be transformed in planta is an endogenous acetolactate synthase (ALS) gene, an Enolpyruvylshikimate Phosphate Synthase Gene (ESPSP) gene, or a male fertility (MS45, MS26 or MSCA1) gene.

As detailed above, under “Definitions”, methods for transforming or manufacturing a plant, plant cell, tissue, organ or material can rely on either the stable integration of a genetic construct or on the transient introduction of a genetic construct or a product thereof according to the present disclosure. The choice of the method, either stable or transient, predominantly is dictated by the effect which is intended to be achieved. Common genetic constructs suitable to be transformed according to the present invention can inter alia be selected from the group consisting of at least one of a viral vector, a CRISPR system encoding construct, an RNAi encoding construct, an miRNA encoding construct, a construct comprising a resistance gene, a construct comprising a fluorescent marker gene, a genetic construct designed to introduce a targeted knock-out of a plant gene or region of interest, a genetic construct designed to introduce a targeted knock-in of a transgene of interest at a plant gene or region of interest, the introduction of a genetic construct mediating a homology-directed repair at a region, where a DNA double-strand break occurred, and a combination thereof.

Other suitable systems for precision genome engineering of eukaryotic genomes, e.g. plant genomes, rely upon homing endonucleases, meganucleases, zinc finger nucleases, and transcription activator-like effector nucleases (TALENs). Said effector nuclease-based systems are likewise compatible with the methods according to the present invention, wherein said methods allow the easy access to a new target locus of a meristematic plant region of interest.

A stable integration might thus be desirable, where a transgenic plant carrying a desired construct of interest, or a part thereof, is stably inserted and the inserted construct or part thereof is inherited to the progeny of a plant cell of interest initially transformed. Said stable integration can take place into any genomic region of the plant, including the nuclear genome as well as the extranuclear genome, including the genome of plastids of a plant cell.

A transient introduction might be desirable, in case a certain effect, e.g. a RNA mediated silencing effect, a targeted manipulation, comprising a knock-in or a knock-out, is desired by the introduction of a genetic construct of interest, or a part thereof, but the construct per se should not be inherited to a progeny of the cell initially transformed.

In another embodiment of the above aspect, the method comprises an additional step (v) comprising determining the functional integration of the genetic construct of interest.

In one embodiment, said determination can take place in situ, i.e. directly in plant by analyzing a transformed at least one meristematic cell or tissue of interest, or a progeny thereof.

In another embodiment, said determination can take place in vitro by explanting or dissecting a transformed at least one meristematic cell or tissue, either as a whole, or a part thereof.

Methods for analyzing a functional integration are known to the person skilled in the art and comprise, but are not limited to polymerase chain reaction (PCR),including inter alia real time quantitative PCR, multiplex PCR, RT-PCR, nested PCR, analytical PCR and the like, microscopy, including bright and dark field microscopy, dispersion staining, phase contrast, fluorescence, confocal, differential interference contrast, deconvolution, electron microscopy, UV microscopy, IR microscopy, scanning probe microscopy, the analysis of plant or plant cell metabolites, RNA analysis, proteome analysis, functional assays for determining a functional integration, e.g. of a marker gene or a transgene of interest, or of a knock-out, Southern-Blot analysis, sequencing, including next generation sequencing, including deep sequencing or multiplex sequencing and the like, and combinations thereof.

In yet another embodiment of the above aspect according to the present invention, the introduction of the genetic construct of interest according to step (iv) of the above aspect is conducted using a means selected from the group consisting of a device suitable for particle bombardment, including a gene gun, including a hand-held gene gun (e.g. Helios® Gene Gun System, BIO-RAD) or a stationary gene gun, transformation, including transformation using Agrobacterium spp. or using a viral vector, microinjection, electroporation, whisker technology, including silicon carbide whisker technology, and transfection, or a combination thereof.

Several methods are known to the skilled person suitable for plant cell transformation. As a prerequisite, said transformation methods all require a plant, plant cell, tissue organ or material to be transformed in a readily accessible state and in a condition so that the respective sample, including at least one cell, is viable. The present invention thus provides a basic method for allowing accessibility of a meristematic cell or tissue of a plant as target structure in a viable state to allow the further manipulation or analysis of the thus exposed target structure in planta.

There is provided a further embodiment, wherein step (ii) of the above aspect comprises the production of an artificially introduced window in the region, where the meristematic tissue of the immature inflorescence is located, preferably in the shoot axis, in particular in the axil, halm, culm or stem of the plant. As it is known to the skilled person in the field of plant biology, the morphology of different plant species can significantly vary and thus the nomenclature for the shoot axis will vary depending on the plant to be characterized.

The term “artificially introduced window” as used herein refers to a purposively introduced lesion being introduced in planta, this means into a growing plant, plant material or seedling, wherein the artificially introduced window is positioned in a region, where meristematic tissue of the immature inflorescence, comprising at least one meristematic cell, is located. As detailed above and as known to the skilled person, meristematic regions or meristematic cells of a developing plant are often surrounded by further tissues which keep the developing meristematic tissue in a protected state. The dimensions of the artificially introduced window naturally can vary depending on the plant, into which the window is introduced. The dimensions (length, width and height) are in a size to fulfill the following conditions: (i) exposition of at least one cell of a meristematic tissue of the immature inflorescence so that said region is preferably macroscopically detectable; and (ii) (for full in planta approaches) the window may be such that the plant is still viable after introduction of the window and can further develop and proceed through further developmental stages, i.e. the window preferably may not remove more than necessary to expose the meristematic tissue comprising at least one meristematic cell. In other embodiments, where the artificially introduced window is created to expose a meristematic tissue of interest to make it accessible for the further explantation/dissection, the window can have greater dimensions to ease the dissection of the material. In this embodiment it is not necessary to take into consideration the further fate of the plant. It is, however, mandatory not to destroy or damage the target structure of interest, i.e. at least one meristematic cell of a meristematic tissue, which is to be cultivated or analyzed further in vitro. The opening of the window can be achieved using physical or mechanical means.

To achieve a suitable artificial window for a selected target plant, first, the developmental stage of the plant is checked by counting the number of leafs. Depending on the genotype, the introduction of the artificial window can be started between the 3 -10 leaf stage, preferably between the 6- and 10-leaf stage. By touching the stem of the plant, the area where the real stem is ending, i.e. the position where the tassel will be located can be determined. Next, a window, preferably with rectangular shape, but not restricted thereto, is cut into the stem with the help of a scalpel or any another mechanical means for opening the stem of the plant of interest exactly at the location where the tassel is likely to be located at. The different leaf sheets are then removed layer by layer until the immature tassel is reached (see e.g. FIG. 5). In the last step, preferably forceps are used to remove the plant material surrounding the tassel to be exposed. As a general rule, the size of the window has to be as small as possible for not disrupting the further development of the plant. The minimum size of the artificial window thus is in the range of approximately 0.5×0.5 cm. Depending on the plant, the window can also be smaller, e.g. approximately 0.25×0.25 cm or even below. A very small window size might possibly complicate the further handling. Using specific equipment, including micromanipulation tools, including inter alia optical tweezers or laser microbeam-assisted tools, the creation of even very small windows can be created.

In another embodiment, the artificial window can have dimensions from around 1 cm×1 cm, or, if a rectangular window is preferred, approximately 1 cm×0.5 cm, approximately 1.5×0.5 cm, approximately 1.5×1 cm or approximately 2 cm×1 cm or any dimensions in between and it can even have larger dimensions depending on the stem architecture of the plant of interest. In principle, there is no maximum size as long as the natural development of the plant is not impeded. There is also no limitation to the geometry of the window, i.e. round or oval windows or the other geometries can be produced as well. Then, a window can have dimensions ranging from a radius of about 0.1 to 2 cm, preferably from a radius of about 0.2 to 1.5 cm, more preferably from a radius of about 0.5 to 1 cm. The specific dimensions will depend on the plant to be windowed and can be determined using practical criteria of handling the respective plant.

In embodiments, where the further cultivation steps are intended to be performed in vitro the window can also be larger, as it is not necessary that the plant can further proliferate after exposition and removal of the meristematic structure, preferably an immature inflorescence, after the window opening.

Preferably, a vertical cut is made from the top of the artificial window towards the leaf whorl in order to help the tassel to grow vertically. After cutting the window, the window is preferably covered back again by one or two cut leaf lids. To prevent the tassel from drying, a piece of wetted material, e.g. cotton (using water or water and a fungicide) can be inserted between the tassel and the lid. The whole artificial window including the lid can then be wrapped with a tissue paper and secured with a metallic twist tie.

In embodiments, where particle bombardment as physical means of transformation is used, the tissue paper, the leaf lid and the cotton are removed and the target structure of interest can then be bombarded before the cotton, the lid and the tissue are again placed back onto the artificial window. To assure a natural development of the exposed tassel tissue, the growth of the tassel is checked daily after transformation. In certain embodiments, it might be suitable to remove that or necrotic tissue to assist the tassel in growing vertically by opening the leaf sheets. In embodiments, where a tissue paper is used, said paper can be removed after around 3-5 days after transformation, including transformation via particle bombardment.

To be able to manipulate at least one meristematic cell of a meristematic tissue, preferably of the immature inflorescence of a plant, it is thus required to expose said meristematic tissue comprising at least one meristematic cell to be manipulated or modified. The artificially introduced window according to the present invention thus has to be made in the region where the target structure of interest is located. Furthermore, in one embodiment the artificially introduced window should be suitable to keep the at least one cell of the meristematic tissue in an exposed state to monitor the further development thereof, if desired, which is preferable for performing intermediate analysis to determine whether a transient introduction or a stable integration according to some embodiments according to the present invention have occurred.

In addition, the artificially introduced window may not destruct the whole plant organism or disturb the further development of the plant, as this would obviate an in planta manufacturing approach according to the present disclosure, wherein the living plant comprising the transformed at least one meristematic cell is further cultivated in planta until a desired developmental stage of the transformed material is achieved. As it is known to the skilled person, the region where an artificially introduced window has to be purposively created to perform the methods according to the present invention can vary depending on the target plant of interest, as the region, where the meristematic tissue of the immature inflorescence is located varies from plant species to plant species. In embodiments, where the further development of the transformed at least one meristematic cell is not proceeded through in an in vitro culture free way, the “artificially introduced window” can have huger dimensions to fully expose a target region of interest, comprising at least one meristematic cell. This is due to the fact that the integrity of the living plant is not a prerequisite in case the further cultivation steps of the transformed material will be conducted in vitro, e.g. via callus regeneration and the like.

In yet another embodiment of this aspect according to the present invention the plant or the plant material is or is part of a monocotyledonous (monocot) plant, or is or is part of a dicotyledonous (dicot) plant.

Monocot and dicot plants, both belonging to the flowering plants or angiosperms, differ mainly in that monocot plants have only one seed leaf (cotyledon) within the seed the shoot is evolving from and dicots have two seed leaves or cotyledons the shoot is evolving from. Both dicot and monocot plants, however, proceed through common stages during plant development for yielding the fully differentiated flowers. Therefore, the methods according to the present invention can be applied for any kind of monocot or dicot plant having a plant architecture, i.e. a three-dimensional organization of the plant body, comprising at least one a meristematic cell of a meristematic tissue of an immature inflorescence and, optionally wherein the plant architecture allows the production of an artificially introduced window in the region, where the meristematic tissue of the immature inflorescence is located.

In a further embodiment according to the above aspect according to the present invention the plant or the plant material is or is part of a plant from a species selected from the group consisting of Hordeum vulgare, Hordeum bulbusom, Sorghum bicolor, Saccharum officinarium, Zea mays, Setaria italica, Oryza minuta, Oryza sativa, Oryza australiensis, Oryza alta, Triticum aestivum, Triticale, Secale cereale, Malus domestica, Brachypodium distachyon, Hordeum marinum, Aegilops tauschii, Daucus glochidiatus, Beta vulgaris, Daucus pusillus, Daucus muricatus, Daucus carota, Eucalyptus grandis, Nicotiana sylvestris, Nicotiana tomentosiformis, Nicotiana tabacum, Solanum lycopersicum, Solanum tuberosum, Coffea canephora, Vitis vinifera, Erythrante guttata, Genlisea aurea, Cucumis sativus, Morus notabilis, Arabidopsis arenosa, Arabidopsis lyrata, Arabidopsis thaliana, Crucihimalaya himalaica, Crucihimalaya wallichii, Cardamine flexuosa, Lepidium virginicum, Capsella bursa pastoris, Olmarabidopsis pumila, Arabis hirsute, Brassica napus, Brassica oeleracia, Brassica rapa, Raphanus sativus, Brassica juncea, Brassica nigra, Eruca vesicaria subsp. sativa, Citrus sinensis, Jatropha curcas, Populus trichocarpa, Medicago truncatula, Cicer yamashitae, Cicer bijugum, Cicer arietinum, Cicer reticulatum, Cicer judaicum, Cajanus cajanifolius, Cajanus scarabaeoides, Phaseolus vulgaris, Glycine max, Gossypium sp., Astragalus sinicus, Lotus japonicas, Torenia foumieri, Allium cepa, Allium fistulosum, Allium sativum, Helianthus annuus, Helianthus tuberosus and Allium tuberosum, or any variety or subspecies belonging to one of the aforementioned plants.

In another embodiment according to the first aspect of the present invention, the immature inflorescence is a male inflorescence (tassel) of a Zea mays plant, or wherein the immature infloresecene is the inflorescence of a Triticum aestivum plant.

The overall architecture of the tassel of a maize plant is depicted in FIG. 1. The tassel, or male inflorescence, in maize is highly branched with long lateral branches at the base of the main spike. Short branches, called spikelet pairs, are produced by the main axis and the long branches. Each spikelet is composed of two reduced leaf-like glumes enclosing two florets (see FIG. 1). Each floret consists of two reduced leaves called the lemma and palea, two lodicules (the remnants of the petals) (Ambrose et al., 2000), three stamens and a tricarpellate gynoecium. In the tassel, the gynoecium aborts resulting in the formation of male florets (Cheng et al., 1983; Irish, 1996). The female inflorescence (the ear shoot) forms from an axillary meristem located in the axil of a leaf five to six nodes below the tassel. The ear does not produce long lateral branches but does produce paired spikelets with paired florets like the tassel. Subsequently, the lower floret and the stamens abort resulting in the formation of single female florets (Cheng et al., 1983; Irish, 1996). Three types of axillary meristems are involved in producing this complex inflorescence (see McSteen et al., 2000 and McSteen and Hake, 2001). The first axillary meristems produced by the inflorescence meristem are the branch meristems. Branch meristems at the base of the tassel produce the long lateral branches while later arising branch meristems (also called spikelet pair primordia) produce two spikelet meristems. Each spikelet meristem forms two glumes and two floral meristems. Subsequently, each floral meristem gives rise to the floral organs. Targeting of meristematic cells of said meristematic tissues is thus favourable to generate a targeted genetic manipulation in the maize male inflorescence. Based on the methods according to the present invention, in one embodiment there is thus provided an approach for directly targeting at least one meristematic cell of a meristematic tissue of the developing tassel of a maize plant in planta which allows the targeted manipulation of this tissue type finally resulting in the reproductive organs and cells. This approach thus paves the way for an in vitro culture free genetic manipulation of this important crop plant within one generation, as the final result of a targeted genetic manipulation can already be obtained in the plant to be manipulated without the need for further cultivation or crossing. The methods according to the present invention are thus suitable for producing chimeric plants or later on transgenic or genetically manipulated plants that will have transformed reproductive cells, tissues or organs.

In yet another embodiment of the present invention, the introduction of the genetic construct of interest as defined in step (iv) according to the above aspect is performed at a developmental stage, either during the stamen initiation process or before spikelets are formed on the male inflorescence.

Given the plant development of monocot and dicot plants, the methods of the present invention specifically targeting at least one meristematic cell of a plant inflorescence as long as meristematic cells are present. For certain approaches, it might be favorable to do the transformation in an earlier developmental state of the inflorescence development to (i) potentially affect a larger cell population of the progenies of the at least one meristematic cell to be transformed and/or (ii) depending on the type of transformation chosen to have less cell layers (glumes, lemma or palea or the epidermis of the anther) surrounding the target tissue or cell. In several dicot plants, where floral meristems can be produced directly from the inflorescence meristem without proceeding through branch and spikelet meristems, it might likewise be of interest to target at least one cell of the inflorescence meristem during an early developmental stage to place the genetic manipulation of interest into as much as possible meristematic cells or progeny thereof.

In a second aspect according to the present invention there is provided a method for manufacturing a transgenic plant comprising the following steps: (i) providing an in planta transformed plant or plant material transformed by a method according to the first aspect of the present invention; (ii) cultivating the plant or plant material under suitable conditions until the developmental stage of maturity of the inflorescence is achieved, (iii) allowing the transformed gamete of the pollen or of the ovule generated from the transformed at least one meristematic cell to fuse to a zygote comprising the genetic construct of interest as transgene; and (iv) developing the transgenic plant from the zygote.

In a third aspect according to the present invention there is provided a method for manufacturing a genetically manipulated plant comprising the following steps: (i) providing a in planta transformed plant or plant material transformed by a method according the first aspect of the present invention detailed above; (ii) cultivating the plant or plant material under suitable conditions until the developmental stage of maturity of the inflorescence is achieved; (iii) allowing the genetically manipulated gamete of the pollen or of the ovule generated from the transformed at least one meristematic cell to fuse to a zygote comprising the targeted genetic manipulation; and (iv) developing the genetically manipulated plant from the zygote.

In one embodiment according to the second or the third aspect of the present invention, the step of cultivation of the plant or plant material under suitable conditions until the developmental stage of maturity of the inflorescence is achieved is performed in vitro culture free.

In another embodiment according to the second or the third aspect of the present invention, the step of cultivation of the plant or plant material under suitable conditions until the developmental stage of maturity of the inflorescence is achieved is performed in vitro. Several protocols are available to the skilled person for producing germinable and viable pollen from in vitro cultured maize tassels as disclosed, for example in Pareddy D R et al. (1992) Maturation of maize pollen in vitro. Plant Cell Rep 11 (10):535-539. doi:10.1007/BF00236273, Stapleton A E et al. (1992) Immature maize spikelets develop and produce pollen in culture. Plant Cell Rep 11 (5-6):248-252 or Pareddy D R et al. (1989) Production of normal, germinable and viable pollen from in vitro-cultured maize tassels. Theor Appl Genet 77 (4):521-526. Those protocols are inter alia based on excision of the tassel, surface sterilization and culture in a media with kinetin to promote tassel growth and maturation. After the spikelets are formed, they a continuous harvest of anthers can be performed. After extrusion, anthers will be desiccated until the pollen comes out. Alternatively, anthers can be dissected and the pollen is shed in liquid medium that is subsequently used to pollinate ears.

“Maturity of the inflorescence” as used herein refers to the state, when the immature inflorescence of a plant comprising at least one meristematic cell has reached a developmental stage, when a mature inflorescence, i.e. a staminate inflorescence (male) or a pistillate inflorescence (female), is achieved and thus a gamete of the pollen (male) or of the ovule (female) or both is present. Said stage of the reproductive phase of a plant is especially important, as the obtained plant material can directly be used for pollination of a further plant or for fertilization with the pollen of another plant.

“Suitable conditions” or “suitable reaction conditions” as referred to herein in the context of the transformation or manufacturing methods according to the present disclosure refer to conditions, which allow both, the growth and development of the plant being transformed or manufactured and the conditions necessary for achieving either stable integration or transient introduction of a genetic construct of interest in the at least one meristematic cell of interest. Conditions to promote plant growth and development, including inter alia temperature, light, water, oxygen, mineral nutrients and soil support, which can vary for different plant species/cultivars can be readily determined by the skilled person in knowledge of the disclosure provided herein. The further suitable conditions to achieve stable integration or transient introduction of a genetic construct of interest depend on the transformation method selected for introduction of the genetic construct, the developmental age of the plant material or plant cell to be transformed and the genetic construct of interest to be introduced. Said suitable conditions can be defined by the skilled person in light of the present disclosure defining the suitable conditions for the methods in combination with exemplary genetic constructs as disclosed and claimed herein.

In one embodiment, where all steps according to the methods of the present invention proceed in vitro culture free, the term “suitable conditions” further can imply that the transformed at least one meristematic cell of the immature inflorescence is kept in a continuing accessible state so that the resulting progeny of the transformed at least one meristematic cell can be directly obtained from the viable plant in a viable state. The term thus implies that, when necessary, an artificially introduced window is monitored and optionally enlarged, as the plant organism by natural phenomena driven by the wound healing capacity of a plant organism might regenerate the plant tissues surrounding the developing meristematic tissue of interest.

In still another aspect according to the present invention there is provided a plant manufactured by the manufacturing methods of the second or third aspect of the present invention, or plant cells, a plant material, or derivatives or a progeny thereof.

In one embodiment of this aspect, the manufactured plant is a monocot plant, in another embodiment the plant is a dicot plant.

In a further embodiment according to this aspect according to the present invention the plant or the plant material is or is part of a plant from a species selected from the group consisting of Hordeum vulgare, Hordeum bulbusom, Sorghum bicolor, Saccharum officinarium, Zea mays, Setaria italica, Oryza minuta, Oryza sativa, Oryza australiensis, Oryza alta, Triticum aestivum, Triticale, Secale cereale, Malus domestica, Brachypodium distachyon, Hordeum marinum, Aegilops tauschii, Daucus glochidiatus, Beta vulgaris, Daucus pusillus, Daucus muricatus, Daucus carota, Eucalyptus grandis, Nicotiana sylvestris, Nicotiana tomentosiformis, Nicotiana tabacum, Solanum lycopersicum, Solanum tuberosum, Coffea canephora, Vitis vinifera, Erythrante guttata, Genlisea aurea, Cucumis sativus, Morus notabilis, Arabidopsis arenosa, Arabidopsis lyrata, Arabidopsis thaliana, Crucihimalaya himalaica, Crucihimalaya wallichii, Cardamine flexuosa, Lepidium virginicum, Capsella bursa pastoris, Olmarabidopsis pumila, Arabis hirsute, Brassica napus, Brassica oeleracia, Brassica rapa, Raphanus sativus, Brassica juncea, Brassica nigra, Eruca vesicaria subsp. sativa, Citrus sinensis, Jatropha curcas, Populus trichocarpa, Medicago truncatula, Cicer yamashitae, Cicer bijugum, Cicer arietinum, Cicer reticulatum, Cicer judaicum, Cajanus cajanifolius, Cajanus scarabaeoides, Phaseolus vulgaris, Glycine max, Gossypium sp., Astragalus sinicus, Lotus japonicas, Torenia foumieri, Allium cepa, Allium fistulosum, Allium sativum, Helianthus annuus, Helianthus tuberosus and Allium tuberosum, or any variety or subspecies belonging to one of the aforementioned plants.

In yet another aspect according to the present invention there is provided a plant, preferably a Zea mays plant, comprising an artificially introduced window in a region, where a meristematic tissue of an immature inflorescence is located, preferably in the axil at the culm of the plant, preferably wherein the plant is a plant in planta transformed by a method according to the first aspect according to the present invention.

In one embodiment, the plant provided according to this aspect is a viable plant. In another aspect the plant is a harvested plant, or a plant cell, tissue, organ or a plant material thereof.

In a further aspect according to the present invention there is provided the use of a plant comprising an artificially introduced window in a region, where a meristematic tissue of an immature inflorescence is located, preferably in the axil at the culm of the plant, preferably wherein the plant is a plant in planta transformed by a method according to the first aspect according to the present invention, or of a plant in planta transformed by a method according to the first aspect according to the present invention, for the manufacture of a transgenic or genetically manipulated plant, plant cell or plant material.

As evident from the various aspects and embodiments according to the present invention, a plant having an artificially introduced window in a region, where a meristematic tissue of an immature inflorescence is located, preferably in the shoot axis, in particular in the axil, halm, culm or stem of the plant, is specifically suitable for a variety of approaches for manipulating the genetic material of at least one meristematic cell of said meristematic tissue, for performing the functional integration of a genetic construct of interest, or for analytical or testing purposes for studying the natural development of the plant. In the natural environment of certain plants, the immature inflorescence meristem is well protected by surrounding cell layers and tissues during early developmental stages. Therefore the provision of a plant having an artificially introduced window exposing the immature inflorescence is useful for the plant transformation and manufacturing methods according to the present invention as well as for further analytical and screening purposes.

The skilled person in the field of plant biotechnology is well aware of the fact that plant organogenesis proceeds differently in various plants, especially when comparing monocot and dicot plants. Therefore, the localization of meristematic cells and tissues which build up the inflorescence meristem or which can develop into the inflorescence meristem is different into the various plant species. Furthermore, the development of floral meristems naturally differs from plant species to plant species. Thus, the floral meristem can either directly develop from the inflorescence meristem, like in dicots like Arabidopsis or Antirrhinum, or the floral meristem can develop from the inflorescence meristem via branch and spikelet meristems, like for maize (see FIG. 1). The person skilled in the art having knowledge about the disclosure of the present invention and the detailed Examples for different plant species disclosed herein can thus transfer the methods disclosed herein to a variety of target plants of interest.

The present invention is further described with reference to the following non-limiting examples.

EXAMPLES

The present invention is further illustrated by the following non limiting examples.

Example 1: Maize Tassel Development

In corn development, the apical meristem differentiates into tassel primordia in the V6 stage (“Vegetative stage 6” or when the plant has 6 leaves) or 25-30 days after germination (Cheng et al. 1983; Abendroth et al. 2011). Therefore, it was tried to target the cells of those immature tassels that will further develop in the anther tissue that will produce pollen. One set of experiments focused on maize plant, namely A188 plants. Already 1 month old plants have developing tassels instead of the classical apical meristems in these plants see (FIGS. 3 and 4). It was observed that the growth of the immature tassel is very fast, going from less than 1 cm to more than 8 in 11 days (FIG. 4). We also looked at the developmental stage of the spikelets and decided that the target age will be between 34 and 37 days after sowing, when the tassel is easy to be detected and exposed but the spikelets are not formed (FIG. 4). This is an important parameter, in case the immature inflorescence is intended to be transformed via a physical method like particle bombardment, as the more cell layers (glumes, lemma or palea and the epidermis of the anther) the particles have to go through to reach pollen producing tissues, the better it is to choose an early developmental stage. For biological transformation approaches, including Agrobacterium transformation, e.g. using viral vectors, there is a greater flexibility in the time range for conducting transformation, as the biological cargo will more easily reach the meristematic target cells of interest without disrupting the surrounding cells and tissue.

Example 2: Exposing the Immature Inflorescence of a Growing Maize Plant

To facilitate the genetic manipulation or the introduction of a genetic construct into the maturing tassel tissue, several methods were analyzed. It was found that a window could artificially be introduced into the region, where the meristematic tissue of the immature inflorescence is located. For maize, this region is located near the axil in the shoot axis, the culm, of the plant. An exemplary plant comprising an artificially introduced window is shown in FIG. 5. The tassel is pushed upwards very fast during plant development. When the tassel is exposed through a window, the natural pathway to grow upwards is disrupted. Sometimes the tassel grows out of the plant or other is trapped between the growing leaf mass, making the plants look abnormal in comparison to a non treated plant (see e.g. FIG. 7 (D to E)), which, however, did not disturb the overall plant and tassel development. For a maize plant, it is thus recommendable to perform a vertical cut from the existing artificially introduced window to the top of the plant to allow the growing tassel to move upwards and mature.

It was observed that in most of the “windowed” plants the tassels are producing pollen, but not all of them develop ears. Usually those plants mature several weeks before a normal A188 maize plant would and they are in general shorter and weaker plants, which however, did not influence the further transformation and analytical experiments as conducted with the exposed material, neither in planta nor in vitro.

Example 3: Transient Transformation of Exposed Immature Tassel with Particle Bombardment In Vitro

One of the questions to be answered by this technology was if the exposed immature tassel tissue is amenable for transformation, especially in planta, but also ex vivo. For that, several experiments were conducted in which the immature tassel was separated from the plant and bombarded in the lab using the bench gene-gun PDS-1000/He (BIO-RAD). Particle bombardment turned out to be a suitable method for all experiments conducted with immature meristematic tissues and isolated cells from several plants including maize, barley and wheat varieties for both in vitro as well as in planta bombardment. Experiments with several test constructs of dsDNA, RNA and proteins as well as virus particles were used in the different settings. During this series of experiments it turned out that the parameters for bombardment are critical to influence, whether a transient or stable expression of a dsDNA construct will be achieved. More heavy bombardment was observed to increase the rate of transient introduction and also can cause a damage of the material bombarded. It is thus necessary to establish the best mode of bombardment individually for each target tissue to be transformed.

Usually, for plant transformation through bombardment, the gene gun known as PDS-1000/He (BIO-RAD) is used, but any comparable device can be used for the presented protocols. The Helios gene-gun is a bench apparatus that relies on helium pressure to discharge the particles to the plant tissue under vacuum conditions. The explants that are bombarded for stable plant transformation purposes necessarily undergo in vitro culture, this is why the explants are usually bombarded on an in vitro culture plate. The Helios gene gun was designed initially to do in situ transformation of animal cells and tissues. The gun can operate without vacuum conditions and at a lower shot pressure, minimizing cell damage (Finer et al. 2000). The use of the Helios gene gun in plant transformation has been limited to transient expression experiments in vitro and no assay could ever be performed in vitro culture free.

The tassel in the present experiments as shown in FIG. 6 is able to transiently express the delivered construct (here: containing a commercially available red fluorescent gene expression cassette). This expression can even be observed in the immature spikelets, what it is an indication that we could be targeting tissue that will produce pollen (FIG. 6).

For this experiment, an immature tassel from a V7-V8 plant was detached and placed into on a petri dish with MS media (MS salts +vitamins, 20 g/L sucrose, 3 g/L phytagel, pH 5.7). Notably, V7 or V8 stage implies that the collar of the 7^(th) or 8^(th) leaf is visible, respectively. The tassel was bombarded once using a red fluorescent protein expressing plasmid coated using standard calcium chloride/spermidine coating on 0,6 pm gold particles. The bombardment was done at 1100 psi, 3¹d shelve from the top of the gene gun. After bombardment the tassel was placed in darkness and the next day, the transient expression was checked under a fluorescence microscope.

Example 4: Stable Transformation of Exposed Immature Tassel with Particle Bombardment In Planta

In the next step the hand held gun Helios was used for bombardment in planta of the exposed immature tassel. Pressures between 100 and 200 psi and from 1 to 6 shots per tassel were used. The distance to the target is the one of the Barrel liner, around 3 cm. It was observed that higher pressures and higher number of bombardments damage the tassel branches. However, transient expression in tassel samples taken 1 day after bombardment (FIG. 7) could be observed. In most of the cases, the bombarded tassels have been able to develop and shed pollen. As tassel tissues like anthers and dry pollen have a strong autofluorescence, a fluorescence detection means can be accomplished and confirmed by further molecular methods, like PCR, including enrichment PCR, or sequencing, including next generation sequencing, including inter alia, deep sequencing, or multiplex sequencing and the like. Bombarded tassels were regularly observed several days after bombardment. Red fluorescent spikelets and glume cells could be observed (FIG. 8) from 7 to 28 days after bombardment, what was indicative of stable transformation events in those bombarded tassels. These results were confirmed by PCR, RT-PCR, sequencing, including deep sequencing or other next generation sequencing approaches, or Southern blot analysis.

As usual set up for analyzing a stable integration event in different target plants was as follows: First, DNA and/or RNA were extracted of different material including tassel, anther or pollen tissue/cells transformed with different constructs encoding a red fluorescent protein. In sum, 89 samples were analyzed via quantitative PCR (qPCR). From the above samples, 20 samples showing a clear, i.e. a very intense, red fluorescent signal were selected. From those 20 samples cDNA was generated including controls without reverse transcriptase to exclude that the later results are not associated with undigested DNA. Out of the 20 samples with positive DNA signal used for the transcription measurement, 6 samples (6.7%) showed a clear transcription and 3 a potential transcription (at the border of what could be clearly measured). In Table 1 below the results for this experiment are listed. Especially in tassel branches very clear signals could be obtained. Also in pollen clear transcription signals have been observed. A further round of experiments changing the parameters for bombardment was done to increase the frequency of stable integration. It was observed that a lower shot pressure and/or choosing a different time point of bombardment also contributed to achieving a stable integration detectable for anther tissue (data not shown).

TABLE 1 Results: Stable integration in 89 tested samples Type of material DNA signal Transcription Tassel Branch 18 of 75  7 of 18 Immature Tassel 1 of 6 1 of 1 Anthers 0 of 3 Pollen 1 of 5 1 of 1

In the next step seeds from tassel bombarded plants were produced and the result of the targeted modification was analyzed in T1 plants. To this end, seeds have been germinated. Then, the leaf tissue was harvested and DNA was extracted therefrom. With this DNA, PCRs on two different transgenes have been performed. The result of the analysis of two different transgenes is shown in FIG. 16 A and B, respectively. As evident from these Figures, sample “41” showed the expected amplificate for both transgene 1 (FIG. 16 A) and trangene 2 (FIG. 16 B). Both positive results have also been verified via qPCR analysis. It was thus possible to detect a positive event in 51 T1 plants by analyzing the seeds from one motherplant.

Example 5: Transformation of Exposed Immature Tassel Tissue

As detailed above, a variety of physical/mechanical as well as biological means for transforming plant cells, tissues, organs or whole plants or parts thereof have been described for introducing genetic material into a plant or plant target structure. After having exposed and thus obtained a tassel tissue according to the disclosure of the present invention, the following methods can be applied to transform this tissue.

Concerning biological means, the tassel tissue or cells thereof can be transformed with Agrobacterium, including Agrobacterium tumefaciens or Agrobacterium rhizogenes mediated transformation. This kind of transformation is well known to the person having skill in the art (see e.g. Jones, H. D. et al., “Review of methodologies and a protocol for the Agrobacterium-mediated transformation of wheat”, plant methods, 2005; or Frame, B. R. et al., “Agrobacterium tumefaciens-mediated transformation of maize embryos using a standard binary vector system”, Plant, 2002). To this end, an Agrobacterium culture comprising a construct of interest is, for example, cultivated over night at 28° C. in fluid Luria Broth medium containing a suitable antibiotic, 10 mM MES and 200 mM ACE. The next day, the over night cultured is centrifuged at 4.400 rpm for 15 min and the supernatant is discarded. The pellet is then again centrifuged for 15 min at 4.400 rpm for 2 min and the remaining supernatant is discarded. The pellet is resuspended (5 ml H2O, 10 mM MES, 10 mM MgCl₂+20 μM ACE). The optical density at 600 nm is adjusted to 1.5. The possibly diluted suspension can then be further used.

Another possibility for transforming immature tassel tissues via biological means is the use of viral vectors. Viral vectors have the advantage that they can be introduced either as DNA or as RNA and to a plant target structure of interest. Furthermore, viral vectors or plant viruses have the capability of spreading into different cells.

For the purpose of the present invention, virus particles, in vitro transcripts of viruses or Agrobacteria carrying a virus encoding T-DNA have been introduced into a plant target structure of interest via filtration (vacuum and non-vacuum). Alternative experiments were carried out using plant sap. To this end, either tobacco or spinach have been infected with the virus of interest to subsequently isolate said virus of interest from the plant sap for infecting another plant target structure, especially tassel tissues from different plants with the plant sap containing the virus.

Despite the biological means of transforming tassel structures of interest, further physical/mechanical means for transformation in addition to particle bombardment were tested.

One suitable method turned out to be microinjection. Microinjection can be used for any kind of meristematic structure tested, preferentially using a microscope with a micromanipulator. Due to the size of certain meristematic structure like tassel or ear meristems, microinjection can be conducted under microscope control or, in case where the target structures are large enough, without microscope assistance. The injection can be conducted, using a variety of methods for a variety of different target molecules to be introduced into a plant target structure of interest including double-stranded plasma DNA, linear double-stranded DNA, RNA and proteins as well as virus particles in liquid solution. These different molecules can be applied with the help of a micro- or nano-needles which assists in injecting the target molecules into the meristematic cell or structure of interest. The target molecules are first coated onto the needle which is then inserted into the meristematic cell or structure of interest.

A further development of this technology is the use of a combination of silicon carbide (SiC) whiskers (e.g. Silar® Silicon Carbide Whisker) and microinjection. To this end, double-stranded plasma DNA, linear double-stranded DNA, RNA, protein or virus particles are precipitated onto the silicion carbide whisker to be injected via a microinjection needle into the meristematic structure or cell of interest. This technique has the advantage that it is not only possible to transfect a single meristematic cell, but there is the possibility to penetrate different cells in parallel due to the spread of the whiskers. Furthermore, the cells get less destructed, as the needle does not have to penetrate into the cell and the whiskers are quite small in size.

Example 6: Optimization of Further Parameters

Depending on the plant species chosen for transformation and depending on transformation methods, several parameters were adjusted to achieve an improved transformation efficiency. In the case of physical transformation methods, especially particle bombardment, a balance between DNA delivery efficiency and damage of the plant tissue has to be determined when a meristematic tissue of interest, either in planta or in vitro as an explants, is transformed. For particle bombardment the parameters tested were helium pressure, number of shots, amount of gold per shot, size of gold particles, amount of DNA per shot, distance to the target, positioning of the gene gun in respect to the tassel or the use of a protective metallic screen. Depending on the target structure to be transformed the shot pressure can be in the range of about 10 to 1500 psi, preferably in the range of about 10 to 1000 psi, more preferably in the range of about 10 to 500 psi, even more preferably in the range of about 50 to 400 psi and most preferably in the range of about 50 to 300 psi. Naturally, the shot pressure will vary depending on the target plant the material is to be bombarded as well as on the result to be achieved (transient versus stable integration of a construct). Preferably, the shot pressure is in the range from 100 to 200 psi. The particle size of the particles, e.g. gold or tungsten, chosen as carrier for particle bombardment can be in the range of about 0.4 to 2 μm as commercially available, yet other dimensions of particles are possible. The particles should not exceed a certain size as this would lead to a further destruction of plant material which could hamper the present protocol if fully conducted in planta/in vivo. Preferably, the particle size is in the range from about 0.6 to 1 μm. A certain degree of damage to the plant tissue to be transformed was well tolerated for in planta approaches and, especially for more mature tassels in maize, provided a better transformation efficiency without hampering the further development of the tassel.

Another factor to be determined is the best developmental age or stage of a plant at the time when transformation is performed. Generally, the more immature the tassel, the easier it will be to target cells that will differentiate in the anther tissue that will produce the pollen. As detailed in FIG. 4, a series of experiments was performed for maize. It was observed that 34 day old plants have a very small tassel difficult to expose, this tassel is very sensitive to bombardment, being damaged very easily. However, hitting one cell in this immature tassel has more chances to become a tissue for pollen production. On the other hand, 37 day old plants were bombarded. In these plants, the main branch of the tassel has already differentiated spikelets. The target in this case are the more undifferentiated branches of the tassel located at the bottom. Those bigger tassels are more resistant to bombardment, but the particles have to go through more layers of cells to reach the sporogenic tissues. Using alternative transformation methods like Agrobacterium transformation of immature tassels or any other transformation method suitable for a plant cell, tissue or organ or a whole plant or material thereof can be applied.

2 approaches were followed in addition to particle bombardment: (1) Agrobacterium injection with the help of a hypodermic needle and (2) opening of an artificial window followed by “spraying” or “coating” the tassel with an Agrobacterium solution. For both approaches, it is desirable to remove the Agrobacteria by known methods a few days after transformation. For tassels derived from a later developmental stage, microinjection might also represent a suitable transformation technique depending on the plant material and the size of the tissue/organ to be transformed.

Another consideration, especially for in planta approaches aiming at the direct harvest of pollen from a transformed plant, is the definition of the best modes to isolate the pollen from the areas transformed, e.g. hit by the bombardment. Four options exist: (1) Harvest the pollen of the whole tassel. (2) Isolate the targeted tassel branches (usually they are shorter and show some signs of damage) by using individual cellophane bags. (3) Cut the non-targeted branches of the tassel and leave only the ones that have been hit. (4) Separate the hit branches for maturation in vitro. All four methods yielded pollen, wherein a genetic manipulation/genetic modification could be detected via PCR. Several methods exist for providing molecular evidence that a transgene of interest was indeed integrated into the genome of a pollen, or for proving the successful transient introduction of a genetic construct and its inherited effect in a pollen. PCR turned out to be the most suitable tool for detection, but any suitable method can be used for screening or analyzing a pollen or likewise an ovule of interest. The pollen of the transformed plant could be directly used to pollinate donor plants to assess the next-generation inheritance of the transgene or the targeted genetic manipulation. The produced pollen from maize could be used to pollinate donor plants. The seeds produced were germinated and the progeny were tested for the presence of the transgene or the targeted genetic manipulation by means of PCR or Southern blotting.

Example 7: Artificial Windowing of Wheat Plants

Several additional experiments have been conducted for testing the applicability of the windowing technology of the present invention with further plants of agronomic interest. One series of experiments was conducted with Triticum aestivum. To this end, the development of wheat plants and the meristem and the tassel tissues thereof has been monitored for different wheat varieties. In comparison to maize, the wheat plant has tillers that are at different developmental stages. Consequently, the age of the plant when the immature ear will be ready cannot that easily be determined as for maize. This can, however, be deduced from the developmental state of each tiller. Furthermore, in wheat, the ear has the male and female flowers. Therefore, when targeting a wheat immature ear, there is the chance of targeting the tissues that will produce ovules and pollen. For the purpose of generating transiently or especially stably transformed plants, this is a huge advantage. As detailed in FIGS. 9 (A-D) the window opening and thus the creation of an artificially introduced window has been conducted for several wheat plants in a comparable manner as done before with maize plants. Fortunately, the meristem in certain wheat varieties is that big that it can be seen by the eye and no microscopy assistance will be needed in comparison to several other plants including sorghum, where the detection of the meristematic tissue can also be accomplished with the help of microscopy assistance.

The thus exposed wheat meristematic tissues as shown in FIG. 9 (A-D) were subsequently transformed either in planta or in vitro. For stable transformation, a particle bombardment approach as detailed above for maize, but using a lower shot pressure in the range of 10 to 500 psi, preferably in the range of 50 to 400 psi. As confirmed by microscopy and further by PCR analysis of the differentiated material from the thus transformed meristem, the stable integration of a fluorescent marker protein into the target cells could be detected.

Example 8: Further Optimization of the Window Opening Process

Initially, once that the window was opened and the immature tassel bombarded as detailed above, the window was closed with a natural “lid”, i.e. a piece of leaf that was originally covering the area, by wrapping it with a tissue paper. This methodology turned out to be suitable, yet might be associated with certain drawbacks depending on the target plant and the target material introduced, since: (1) the wounded tissue could be very prone to get fungal infections, or (2) as the part of the tassel or the immature inflorescence is exposed to air (the bombarded or otherwise transformed or transfected part). This exposition to air is drying the tassel branches or the immature inflorescence so that a further development in planta or in vitro might not be possible. If not working under highly controlled conditions, which is not always feasible when performing high-throughput experiments, these points might be of importance so that optimized conditions have been elaborated. To avoid this from happening a wetted cotton pad or tissue was placed covering the bombarded or otherwise transformed tassel or immature inflorescence first. This measurement can avoid the drying, but, if not adequately controlled, can promote fungal infection. An improvement to those problems is the application of waxes or Vaseline-type substances to the wounded area. We have tested pure Vaseline, different mixes of natural wax and Vaseline, and commercial products available in garden shops to heal wounds in trees (professional wound covering substances). These practices are common in the grafting technologies. Additionally, instead of wrapping the wound with a paper tissue, professional grafting tape or parafilm have been used, improving sealing the wound and protecting it from fungal infections and dehydration. When tissue paper was used it could be observed that, after bombardment, few bombarded tassels for maize as target plant developed into maturity or that they were covered in fungi and only some branches ultimately produced viable pollen. With the Vaseline/wax treatments in most of the experiments tassels are developed to maturity. Success rates of 75% or more could be observed for this strategy.

Example 9: Substitution of Bombardment with Agrobacteria Injection

First, the susceptibility of immature tassel tissues for Agrobacterium (Ab) infection was tested. A construct expressing a red fluorescent was used to transform an immature tassel cut and separated from the plant. The plants were on a V6-V7 stage and the tassels were between 2 and 3 cm long. The bacteria were grown to an OD₆₀₀ of 1.0 and the tassel was inoculated for 10 minutes. Two days after infection the red fluorescence was monitored. Several red fluorescent spots were observed in the tassel, confirming the suitability of this method for plant transformation (FIG. 10). Then, plants in a V6-V7 stage were used to open a window and leave the immature tassel tissues exposed. Agrobacterium carrying a red fluorescent protein expressing construct was injected at an OD of 0.7. Around 100-200 μl of the bacteria were injected per tassel. The procedure of introducing a window and the subsequent injection with Ab is shown in FIG. 11. Subsequently, the tassels were covered with the Vaseline/parafilm method and the red fluorescence was monitored 2 days after infection. In order to avoid Agrobacterium overgrowth, 2 to 7 days after injection a solution of antibiotics (e.g. timentin, carbenicillin, cefotaxime, etc.) was applied to the infected tissue. The treated tassels were grown to maturity and a self-pollination was performed. In the T1 generation the molecular analyses were performed to confirm the stable or transient transformation event of interest. These experiments could confirm that an in planta method can be performed to transform meristematic cells of a maize plant without hampering the further development of the thus modified immature inflorescence so that pollen can directly be obtained from a modified plant without cumbersome (in vitro) cultivation steps.

Example 10: Applicability to Different Maize Genotypes

The above experiments have been performed in different corn genotypes: A188, Va35 and A632. In all of them it was possible to expose the tassel, perform the bombardment and obtain a mature pollen producing tassel. For each genotype the vegetative stage in which the tassel is ready to be transformed naturally varies, for example, in A188 the stage is V6-V7, while A632 have to be targeted between V7 and V9. The optimum stage can be easily determined after corresponding pre-experiments. In any case, the method is showing to be applicable to a variety of maize genotypes, showing the genotype independence.

Example 11: Embryo Meristem Bombardment

To further optimize the above-identified methods, a so called embryo meristem bombardment method was established which allows to directly obtain a plant or plant material carrying a desired targeted genomic modification from an immature embryo without time consuming cell culture steps being prone to contamination. To this end, bombardment of meristem regions of immature embryos have been run in a pipeline mode for two different genotypes: A188 and A632, what shows the genotype independecy of the transformation method. Around 100 embryos (FIG. 12 A) were bombarded with CRISPR/Cas9 constructs along with a red fluorescent protein expressing plasmid. One day after bombardment the red fluorescence was monitored (FIG. 12 B) and the embryos that did not show fluorescence in the meristem area were discarded. After embryos germinated, a 25% of the germinated plantlets were analyzed at the molecular level. The rest was grown to maturity in the greenhouse. Once the plants reached the reproductive stage a sample of the tassel and the ear were taken and analyzed for transgene. If a positive outcome was found, the plant was self-pollinated and the progeny were analyzed. The produced plants showed sometimes a slow growth and a stunted phenotype compared to the wildtype, however, most of them were fertile and produced seeds. It could thus be demonstrated that this kind of transformation represents a highly efficient method to introduce a genetic construct of interest in a quick and reliable way in a meristematic plant target structure of interest to produce a heritable modification.

Example 12: Meristem Access in Different Plants

As detailed above, it was an objective to edit the genome of a plant by applying the genetic modification tools in planta both in a stable, but preferably also in a transient way. Traditional plant transformation methods rely on the transformation of few cells which after reprogramming in vitro can regenerate into a whole plant. This reprogramming usually proceeds through organogenesis (e.g. shoot multiplication) or embryogenesis (somatic embryo production). Therefore, when the delivery of genome modifying tools has to be done “in planta” as presented herein, there is provided a direct and efficient way to target cells that will immediately develop in a reproductive organ (female flower, male flower, inflorescence, pollen, ovules). The such modified reproductive organ will then produce progeny that will inherit a genetic modification of interest. An alternative to target a variety of different crop plants is the transformation or targeting of microspores (immature pollen) or pollen grains. Those tissues, if transformed, could be used to pollinate and obtain modified progeny. There are few examples in literature, most of them related to bombardment and transient expression analysis of the delivered genes (Twell et al. 1989, Obert et al. 2008). However, the technology will be developed to mature those targeted microspores or pollen to pollinate and recover modified progeny. The method for this technology is very similar across crops. Targeting of microspores is done directly in immature anthers or by releasing the microspores in a culture media. This targeting can be done for example through bombardment or microinjection. It was successfully used to produce transgenic tobacco plants (Touraev et al. 1997) and cotton (Gounaris et al. 2005). As for targeting mature pollen, the pollen can be freshly collected and treated through bombardment or sonication (reviewed in Eapen (2011)) and immediately perform pollination of for example maize ears (Horikawa et al. 1997). The progeny can be then analyzed for the presence of transgenes or editing events.

Sugar Beet

Immature embryos can be obtained as described in Zhang et al. (2008). Flower spikes were obtained from bolted plants grown in the greenhouse 14 days after anthesis. They were sterilised in 30% Domestos for 30 min. Immature embryos (lEs) were isolated from flower stalks and subsequently cultivated for 4 weeks on solid MS media supplemented with various plant growth regulators. A representative embryo is shown in FIG. 13. In those immature embryos the shoot apical meristem can be targeted directly (like with microinjection) using a microscope to detect the meristem area or with more random targeting technologies like bombardment. Once the meristem is hit, a time for recovery and embryo maturation has to be done. This embryo maturation takes place in an incubator in the dark in a range of temperatures from 20-30° C. The maturation period takes place from 1 to 4 weeks. Once the embryo is matured and starts to germinate it is moved to a MS solid media in the light for the plantlets to develop. When those plantlets are robust enough they are transferred to soil with an acclimatation period of 1 to 4 weeks. Those plants are then grown normally and the progeny is analyzed.

Targeting the mature embryo of sugar beet requires removing the hard pericarp of the seed (Hermann et al. 2007). The embryo is located in the middle and the shoot apical meristem is reachable. Before removing the pericarp a sterilization of the seed using bleach and ethanol has to be done. Then, with the help of scalpels or other sharp tools the pericarp is removed and the embryo is exposed. This embryo is then placed in a media suitable for the specific method of transformation. The meristem of the mature embryo can be directly targeted using a microscope or the whole embryo can be subjected to transformation, randomly reaching the meristem area. After a period of resting of 1 to 10 days in an incubator in the dark (20-30° C.) the embryo germinates and the plantlet is potted. The sugar beet plant is then grown to maturity and the progeny is analyzed.

Shoot Meristem

The shoot meristem in sugar beet plantlets can be targeted by performing cuts in the meristem area (Artschwager 1926). These types of shoot meristems have been targeted already. To this end sugarbeet apices were used as targets for particle bombardment with a microtargeting device. Before examining gene expression, particle penetration experiments were carried out. Transient GUS expression was detected within the first and second cell layers of the meristem. Dividing cells with GUS activity demonstrated that cells survived the bombardment procedure (see Mahn et al. 1995). Additionally, it has been suggested that the incubation of cut meristems with disarmed Agrobacterium strains might be a method in B. vulgaris to introduce genetic information and to result in transgenic plants (chimaeras) (Krens et al. 1988).

Targeting the meristem of a plantlet is done as follows: the sugar beet seeds are surface sterilized and germinated in filter paper or solid media. This germination can take place in the dark or in light conditions. The plantlets are then prepared for meristem targeting. One way is to make some cuts in the meristem area in order to facilitate the access of different transformation methods (e.g. microinjection, Agrobacterium). Those cuts can be of around 1-10 mm and can be in a horizontal or vertical to the axis of the plant. Additionally, the complete meristem can be exposed by peeling all the leaf material that is covering it. Those exposed meristems can be treated with substances that can diminish the physical damage and increase survival, like antioxidants. Once the method of transformation has been applied, the wound can be closed by physical methods (covering with a tissue, parafilm or other materials), or it can be left open. The treated plant must recover from the injury and restart the vegetative growth. For this, the plantlets are cultivated in the dark for 1-10 days and then transferred to light for normal growth. The plants are then transferred to the greenhouse until flowering, and the progeny analyzed.

To target the meristem of an adult plant, plants in different vegetative stages can be used. Following a similar approach, the main shoot meristem or the axillary ones can be exposed by removing the leaf material that covers with the help of a scalpel. Once the meristems are reached, the treated plant is cultured until it flowers, and the progeny is analyzed. For this series of experiments, a bombardment of meristematic tissue coming from plantlets multiplied in vitro was done. The leaf material was removed until the meristematic tissue was exposed. The meristematic areas were then cut vertically and exposed the cut area up or vertically without any cutting. After bombardment with the bench gene gun the explants were left in vitro. One day after bombardment cells showing presence of beta-glucuronidase activity were found, confirming that the meristematic area is receptive to bombardment (FIG. 14).

Targeting Floral Organs

In addition, the inflorescence of sugar beet can be targeted while it is developing, before flower formation or directly in the immature flowers. The flowers are then left to mature and after pollination, the seeds are harvested and the progeny is analyzed. In Beta vulgaris, the inflorescence is compound and consists of an indeterminate main axis with many closed, dichasial and sympodially branched inflorescence units. The terminal flower of each inflorescence unit and one of the lateral flowers fuse at a later developmental stage. Floral parts originate starting from the outer whorl of five asynchronically developed tepals towards the gynoecium. The five stamen primordia originate free from each other, and they are raised in the course of floral development by the formation of an (intra-)staminal ring from an annular intercalary meristem (Olvera et al. 2008).

Wheat Shoot Meristem in Embryos

Immature kernels are collected from immature ears 5 to 20 days after anthesis. Those kernels are surface sterilized using bleach and ethanol. The immature embryos are then extracted with a scalpel under a microscope. Those embryos have the meristem exposed at different degrees. Those meristems are then targeted by the different transformation methodologies like described in Sautter et al. (1995). Once the embryos have been targeted, for example with a construct encoding or providing a red fluoprescent protein (see FIG. 15 A and B), they are cultured in an embryo maturation media like described in Matthys-Rochon et al. (1998). The germinating plantlets were then acclimated and transferred to the greenhouse. The plants are grown to maturity and the progeny analyzed.

Shoot Meristem in Plantlets

Seeds of wheat are germinated in soil or solid media and the meristem of the plantlets can be targeted following the protocol described in (Sautter et al. 1995, Sautter et al. 1995). Briefly, for the production of vegetative shoot apical meristems, seeds can be sterilized by soaking in 70% ethanol for 2 min, followed by sodium hypochloride and four water rinses. The sterile seeds can be sown in glass tubes on MS medium supplemented with 100 mg/L cefotaxime, 2% sucrose and 0.8% Difco agar. Shoot apical meristems from 6- to 10-day-old plantlets can be exposed by removal of the coleoptiles and the first three to five leaves. Roots are then trimmed to about 5 mm. The explants are then placed on MS-basal medium supplemented with different sucrose concentrations (optimum: 10%), and 0.8% agarose. After particle delivery or transformation, the explants can be transferred to MS-agarose and for prolonged culture onto MS-agar 5 days following bombardment.

Targeting Floral Organs

The shoot meristem in wheat differentiates into immature ears very early in the tiller development, few weeks after sowing. Those immature ears can be found at the bottom of the tiller (see FIG. 9 A). These immature ears can be targeted by cutting the leave sheaths of the tiller, opening a window. Through this window the ears can be accessed by different transformation techniques. After transformation the wound is sealed and the plant is let to grow to maturity and the seeds are harvested (cf. FIG. 9 B and D). The progeny is analyzed. As an alternative, the immature spikelets can be detached from the inflorescence, surface sterilized and targeted in vitro. The maturation and seed production can be done in vitro with modifications to the protocol described by Barnabas et al. (1992).

Additionally it has been shown that targeting those immature inflorescences using a gene gun is possible (Leduc et al. 1994, Sautter et al. 1995). After bombardment, cells in the immature inflorescence were expressing reporter genes and undergo cell division.

Oilseed Rape Shoot Meristem in Embryos

Shoot apical meristem (SAM) in embryos is can be already detected in the “heart” stage of development. The globular-shape embryo stage is reached approximately six days after flowering (DAF), the heart-shape embryo stage is reached after eight days, and by day 14 the seed enters the cotyledon stage” (Huang et al. 2009).

To target the meristem of immature embryos of oilseed rape for the purpose of the above methods, pods are harvested 7-21 days after flowering. The pods are opened and the immature seeds are surface sterilized with bleach and ethanol. The embryos in heart or cotyledon stage are then extracted and cultured in an embryo maturation media. In this media the embryos are positioned to be targeted by different delivery methods. After delivery, the embryos are cultured in the dark for 2-15 days until they mature and start to germinate. The germinating plants are transferred to the greenhouse, grown to maturity and the seeds are harvested. The next generation seeds are sown, and leaf samples are taken to check for genome editing events.

Shoot Meristem in Plantlets

The shoot meristems in oilseed rape plants were targeted in the plantlet stage right after germination or when the plant had 2-8 true leaves. The leaf primordia covering the meristem were peeled with the help of a scalpel leaving the meristem exposed. The meristems can be treated with substances like antioxidants that will improve the survival of the manipulated plants. Different delivery technologies are then applied to the meristem. After delivery, the plant is left to recover. Once the plant continues developing normally, the plant is grown to maturity and the progeny is analyzed for the presence of the introduced targeted genomic modification of interest.

Targeting Floral Organs

The flowers in an oilseed rape inflorescence are continuously produced. At the tip of the floral racemes new flowers are produced. When targeting the floral organs of oilseed rape, two approaches were followed. In the first one, immature individual flowers were opened in situ and reproductive tissues were targeted. After the targeting, removal of non-treated flowers and pods was performed and the inflorescence was covered to promote self-pollination. The seeds were harvested and the progeny analyzed for editing events. In the second approach, all differentiated flowers were carefully removed from the raceme and the floral meristem was left exposed. This meristem was then treated with different delivery systems. The meristem was then covered and let to continue the normal development. All the pods produced from this treated floral meristem were harvested and the progeny tested for editing events.

Soybean Shoot Meristem in Embryos

The shoot meristem of embryos was be exposed and transformed as described in (McCabe et al. 1988): Mature seeds of commercial Brazilian soybean cultivars (BR-16, Doko RC, BR-91 and Conquista) were surface-sterilized in 70% ethanol for 1 min followed by immersion in 1% sodium hypochlorite for 20 min. and then rinsed three times in sterile distilled water. The seeds were soaked in distilled water for 18-20 h. The embryonic axes were excised from seeds, and the apical meristems were exposed by removing the primary leaves. The embryonic axes were positioned in the bombardment medium (BM: MS (Murashige and Skoog 1962) basal salts medium, 3% sucrose and 0.8% phytagel Sigma, pH 5.7) with the apical region directed upwards in 5-cm culture dishes containing 12 ml culture medium. As soon as the embryonic axes-derived shoots were 2-3 cm in length, a 1-mm-long section could be removed from the base of each leaf for analysis of GUS (beta-glucoronidase) expression (McCabe et al. 1988). The shoots expressing the exogenous DNA could be individually transferred to a plastic pot containing 0.2 dm³ of autoclaved fertilized soil:vermiculite (1:1), covered with a plastic bag sealed with a rubber band and maintained in a greenhouse. After 1 week, the rubber band was removed. After an additional week the plastic bag was also removed. As soon as the acclimatized plantlets reached approximately 10 cm in length they could be transferred to a pot containing 5 dm³ of fertilized soil and allowed to set seeds (McCabe et al. 1988). Once the plantlets are grown, leaf samples can be taken for analyses and the plants are grown to maturity to analyze the progeny to verify the presence of a genomic modification introduced in a targeted way.

Alternatively, the meristem of immature embryos was targeted. For this, pods 5 to 20 days after flowering were harvested and embryos between the heart or cotyledonary stage were extracted with the help of a scalpel and forceps. Those embryos were cultured on an embryo maturation media and the shoot apical meristem was targeted using different delivery methods. The embryos were then cultured in darkness for 1 to 10 weeks until complete maturation as described in Buchheim et al. (1989) and were cultured in light for embryo germination. The produced plantlets were cultured until maturity in the greenhouse. The seeds can be harvested and the progeny can be analyzed on a molecular level.

Shoot Meristem in Plantlets

The targeting of the shoot meristem of germinating plantlets of soybean was done as described in (Chee et al. 1989). Briefly, seeds of Glycine max L. Merr. (cv A0949) were sterilized by soaking in a 15% Clorox solution for 15 min, followed by several rinses with sterile distilled water. Seeds were germinated on sterile moistened paper towels in Petri dishes for 18 to 24 h, at 26° C., in the dark. Seed coats were removed, and one of the two cotyledons of each germinated seed was removed and the half seed with the plumule, cotyledonary node, and adjacent cotyledon tissues attached was inoculated with overnight liquid cultures (A600=0.5) of avirulent Agrobacterium strain C58Z707 which contained the binary plasmid pGA482G. In this case, they did Agrobacterium inoculation, but this transformation could be substituted by other delivery methods. Another approach that was followed was the one described in (Chowrira et al. 1995). In this case, the terminal bud of plantlets 7-10 days old was exposed by removing the surrounding leaf tissue. Foreign DNA was injected with a syringe containing lipofectin, and after, the meristem was electroporated. The plants were grown to maturity with no selection and chimeric plants were obtained. The progeny was analyzed.

Targeting Floral Organs

The access to the stigmas of pollinated flowers of soybean was done as described in Shou et al. (2002). Briefly, all experiments were performed in the late afternoon on flowers that had been self-pollinated naturally that morning. Two wing petals and one keel petal were removed to expose the stigmas of soybean flowers. Stigmas were severed at the boundary between the ovary and stigma, and 10 μL of the plasmid DNA (concentrations of 25, 80, 100, or 150 μg/mL) was applied to the exposed stigma. The DNA solution was retained by the banner petal and calyx. Treated flowers were identified by tagging, and untreated flowers and buds at the same node were removed. The pods that developed from the treated flowers were harvested individually.

Alternatively, the floral meristem of soybean inflorescence was targeted before the raceme developed by removing the primordia that covers it, or when the flowers started to develop in the raceme. This exposure was done by excision of the primordia using a scalpel. Once the flower meristem was exposed the delivery system was applied and the raceme was covered with a paper bag. After the inflorescence developed and the self-pollination happened, the pods coming from the treated plants can then be harvested, the seeds can be processed and the progeny can be tested for genome editing events.

Cotton Shoot Meristem in Embryos

The experiments for targeting the meristem in embryos of cotton were done as described in Aragão et al. (2005). Briefly, seeds (var. 7MH, CD-401, Antares and ITA94) were harvested manually, and the lint was removed by acid treatment. Concentrated sulfuric acid was added to seeds (3 mL/g seeds) and vigorously mixed using a glass rod for 1 min. Seeds were immediately transferred to 5 L of water, rinsed three times with distilled water and dried on paper towel. Mature seeds were surface sterilized in 70% ethanol for 1 min followed by 2.5% calcium hypochlorite for 10 min, and rinsed three times in sterile distilled water. Next, seeds were soaked in distilled water for 24 h, following which the water was removed with seeds allowed to germinate in the dark for 16 h at room temperature. Embryonic axes were excised from seeds, and apical meristems were exposed by removing the cotyledons. Explants were transferred to MS medium containing 3% glucose, 5 mg/L benzylaminopurine (BAP), 0.8% phytagel (Sigma). The pH was adjusted to 5.7 prior to autoclaving. Embryonic axes were prepared as described above and positioned in the bombardment medium (MS basal salts medium, 3% glucose, 5 mg/L BAP and 0.8% phytagel Sigma, pH 5.7) with the apical region directed upwards in 5 cm culture dishes containing 12 mL culture medium. After this, the delivery methods were applied. The treated apical meristems were cultured in the dark and let to recover. The meristems that showed growth were transferred to a light growth chamber and afterwards, plantlets were transferred to the greenhouse to maturity. The progeny was analyzed.

Alternatively, the meristems of embryos were transformed as described in Rajasekaran (2013).

In another approach the meristems of immature embryos in the heart stage were targeted. For this the embryos were matured in vitro following the protocol described by Mauney (1961), meaning the culture medium used was composed of White's nutrient mixture with all ingredients at five times the usual concentration and supplements of 40 mg/l adenine sulfate, 250 mg/l casein hydrolysate, 150 ml/l coconut milk, and 7 gm/l NaCl. The medium was hardened with 8 gm/l Bacto-agar and 20 gm/l sucrose was included as carbohydrate source. 2. The principal feature of this medium in the success of the culture method was the adjustment of the osmotic pressure to a high level by the inclusion of the 7 gm/l NaCl. After the embryos had grown on this high-osmotic-pressure medium for 3-4 weeks, they were transferred to a medium of intermediate osmotic pressure (3 gm/l NaCl replacing the 7 gm/l) and then, after a further growth period of 2 weeks, to a medium containing no NaCl. The successfully cultured embryos germinated on the last medium and were potted in soil.

Shoot Meristem in Plantlets

In the first approach, we transformed cotton plantlet meristem as described in Zapata et al. (1999). Briefly, seeds were surface-sterilized with concentrated sulfuric acid (1 h), 50% Clorox (1 h) in a rotary shaker at 50 rpm and rinsed at least three times with sterile double-distilled water. The seeds were then placed on 0.15% (WN) gelrite-solidified medium, pH 5.7, containing the Murashige and Skoog (MS) inorganic salts (Murashige and Skoog 1962), and 2% of sucrose in order to promote germination. Seeds were incubated at 28° C. in the dark for approximately 3-4 days. The shoot apex was isolated with the aid of a dissecting microscope and was then placed in MS inorganic salts (Murashige and Skoog 1962) supplemented with 100 mg/l of myo-inositol, 0.5 mg/l of thiamine) HCl, 0.5 mg/l of nicotinic acid, 0.5 mg/l of pyridoxine) HCl, 3% sucrose, and 0.15% (W/V) gelrite at pH 5.7. After the isolation, the delivery systems were applied and the shoots were transferred to the shoot-apex medium described above. The surviving shoot apices were transferred to fresh medium. After this period the shoot apices that survived were transferred to the same medium without kanamycin so that roots could develop. Rooted plants (TO) were then transferred to soil and grown to maturity in a greenhouse. The progeny could then be analyzed.

Alternatively, meristems were transformed as described in Keshamma et al. (2008). Briefly, seeds of a breeding line of cotton viz., NC-71 were soaked overnight in distilled water and were surface sterilized first with 1% Bavastin for 10 mins and later with 0.1% HgCl₂ for few seconds and washed thoroughly with distilled water after treatment with each sterilant. The seeds were later allowed to germinate on petri plates at 30 ° C. in dark. Two-day old seedlings can then be taken as explants. The seedlings with just emerging plumule were infected by separating the cotyledons without damaging them such that the meristem is visible. Then the delivery method of choice was applied and the seedlings were transferred to autoclaved soilrite (vermiculite equivalent) moistened with water for germination under aseptic conditions in the growth room in wide mouth capped glass jars of 300 ml capacity, 5 seedlings per jar. After 5 to 6 days, the seedlings were transferred to soilrite in pots and were allowed to grow under growth room conditions for at least 10 days before they were transferred to the greenhouse. The plants were grown to maturity and the progeny was analyzed. In another approach cotton meristems were transformed following the protocol described in McCabe et al. (1993).

Targeting Floral Organs

In another approach the flowers of cotton were targeted following the procedure described in Gounaris et al. (2005). For this, cotton plants of the cultivar Christina were used for transformation. Flowers to be used as pollen receptors in pollination experiments were emasculated two days before the anticipated time of anther dehiscence. In the morning of the day when pollination was to be performed, donor flowers with intact stamens were collected 1-2 hours after dehiscence. Each donor flower was the treated with the delivery methods. These inflorescences were used to pollinate 15-20 emasculated receptor flowers. The pollinated flowers were allowed to develop and produce seeds. The progeny can then be analyzed by the method of choice.

In another approach, flower meristems or immature flowers were targeted. For this, the meristem forming the fruiting branch, the fruiting bud and the immature flower were exposed by removing the primordia and the leaf like bracts. The delivery method was then applied to the exposed meristematic tissues. After this, the treated zones were covered and let to grow. The newly formed flowers were covered and the fruits were harvested. The progeny can then be analyzed by means of molecular biology or, if pertinent, by analysis of the phenotype.

Rice Shoot Meristem in Embryos

Following the protocol of Naseri (Naseri et al. 2014), Rice (O. sativa var Hashemi) seeds were sterilized by soaking in 90% ethanol (1 min) and washed with water three times. Sterile seeds were placed on wet cotton at 22° C. for two days. At this stage, A. tumefaciens was inoculated into embryonic apical meristem of the soaked seeds, a region on the seed surface where a shoot would later emerge and was pierced twice up to depth of about 1 to 1.5 mm with a needle (φ 0.70 mm) dipped in the A. tumefaciens inoculums. The inoculated seeds were then placed on filter papers on wet perlite in flasks covered with aluminum foil and incubated at 23° C. in dark for nine days, during which 70 to 75% of inoculated seeds germinated to seedlings. In order to eliminate A. tumefaciens, the seedlings were immersed, at room temperature, in an aqueous solution (1000 ppm) of cefotaxime for 1 h. Subsequently, seedlings were transformed to basins containing Yoshida solution for rooting. Finally, seedlings were planted in pots and grown to maturation (T0) under non-sterile conditions and allowed to pollinate naturally to set seeds (T1).

In another approach, the meristems of immature embryos were targeted. Immature seeds were harvested 3-12 days after pollination. The immature embryos were placed in maturation media (Ko et al. 1983) and the delivery system(s) of choice was/were applied. The embryos were matured as described in Ko et al. (1983). The germinating plantlets were acclimated in the greenhouse and grown to maturity. The seeds were harvested and the progeny was tested.

Shoot Meristem in Plantlets

The meristem of rice plantlets were targeted as described in Muniz de Péadua et al. (2001). Briefly, rice seeds were surface sterilized and germinated in vitro. After approximately 4 days the shoot apices were excised from the distal part of the first internode of the epicotyl and the coleoptile was removed. Once the meristem was exposed the delivery methods were applied. The growing plant was then let to root. The recovered plantlets were transferred to the greenhouse to grow until maturity and the progeny was analyzed.

Targeting Floral Organs

Immature rice spikelets were targeted as described in Rod-in et al. (2014). Briefly, the inflorescences at stage 51 (the beginning of panicle emergence: tip of inflorescence emerged from sheath) according to the BBCH-scale (Lancashire et al., 1991) were used. The tips of selected rice spikelets were cut off before the inflorescence was treated with the delivery method of choice. Each treated inflorescence was covered with a plastic bag (to maintain humidity) at 25° C. for 3 d. The seeds from the treated spikelets were harvested and the progeny was analyzed.

In another approach the floral meristems were exposed by dissecting with a scalpel de surrounding tissue. The exposed meristems were then treated with the delivery methods. After the targeting the area was covered to keep the moisture. The growing inflorescence tissues were bagged. Once the seeds were harvested, the progeny was analyzed. In yet another approach, the immature spikelet was targeted. In the immature rice inflorescence the immature spikelets were targeted before differentiation or were opened by cutting the outer primordia. The delivery method was then applied. The treated spikelets were covered and let to mature. The seeds coming from these flowers were harvested and the progeny was tested for editing events (Itoh et al., 2005).

Example 13: Further Delivery Methods for Transient Introduction

Due to regulatory and safety reasons, controllable transient delivery methods for plant genome editing are getting more and more important. Therefore, several specific transient delivery methodologies have been evaluated and elaborated that can be applied to the meristems in accordance with the in planta transformation approach of the present invention. Naturally, delivery methods are tissue dependent and thus some of the following methods can be applied generically and others are specific to certain types of tissues (pollen, meristem, flower, etc.).

Cas9 Protein

Cas9 protein was purchased from New England Biolabs, PNA BIO, ToolGen, LDBIOPHARMA or ABM, or was purified as described in Liu et al. (2015).

In Vitro Transcription of sgRNAs

In vitro transcription was done as described by Zuris et al. (2015). Briefly, linear DNA fragments containing the T7 promoter binding site followed by the 20-bp sgRNA target sequence were transcribed in vitro using the T7 High Yield RNA Synthesis Kit (NEB) according to the manufacturer's instructions. In vitro transcribed RNA was precipitated with ethanol and purified by gel electrophoresis on a Criterion 10% polyacrylamide TBE-Urea gel (Bio-Rad). Excised gel fragments were extracted in 420 μl of 300 mM NaCl overnight on a rocking surface at 4° C. Gel-purified sgRNA was precipitated with ethanol and redissolved in water, and sgRNA concentration was finally quantified by UV absorbance and snap-frozen at −80° C.

Alternatively, gRNAs were obtained as described in Kim et al. (2014). Briefly, RNA was in vitro transcribed through run-off reactions by T7 RNA polymerase using the MEGAshortscript T7 kit (Ambion) according to the manufacturer's manual. Templates for sgRNA or crRNA were generated by annealing and extension of two complementary oligonuceotides. Transcribed RNA was purified by phenol:chloroform extraction, chloroform extraction, and ethanol precipitation. Purified RNA was quantified by spectrometry.

Alternatively, the protocol described in (Ramakrishna et al. 2014) was followed. Namely, RNA was in vitro transcribed through run-off reactions by T7 RNA polymerase. Templates for sgRNA transcription were generated by annealing and extension of two complementary oligonucleotides (Supplemental Table 1). Transcribed RNA was resolved on an 8% denaturing urea-PAGE gel. RNA was recovered in nuclease-free water followed by phenol:chloroform extraction, chloroform extraction, and ethanol precipitation. Purified RNA was quantified by spectrometry.

Complexing Cas9 Protein and gRNA

Like described in Zuris et al. (2015). for the delivery of Cas9:sgRNA complexes, 1 μl of 200 μM Cas9 protein was mixed with 2 μl of 50 μM sgRNA and incubated for 5 min at room temperature before mixing with 3 μl of either RNAiMAX or Lipofectamine 2000 and incubating for an additional 30 min before injection. Alternatively, the complexing was done as described by Kim et al. (2014). Namely, Cas9 protein (4.5-45 mg) was premixed with in vitro transcribed sgRNA (6-60 mg). Cas9 protein in storage buffer (20 mM HEPES pH 7.5, 150 mM KCl, 1 mM DTT, and 10% glycerol) was mixed with sgRNA dissolved in nuclease-free water and incubated for 10 min at room temperature.

Agrobacterium Pollen Transformation

The pollen transformation through Agrobacterium was performed according to Li et al. (2004). Briefly, pollen grains were collected from flowers with newly dehisced anthers. An aliquot of Agrobacterium tumefaciens suspension was placed in a sterile 1.5 mL centrifuge tube and centrifuged for 10 min at 3000 rpm. The pellet was resuspended in the pollen germination medium with approximately 50 mg of pollen per milliliter and vacuum (-80 Pa) was applied for 30 min and then slowly released. The suspension was subsequently centrifuged at 3000 rpm for 5 min and the pellets (pollen) immediately used for pollination.

Shoot Apical Meristem Transformation

Meristems of germinating seedlings were transformed based on the protocol described by Chee et al. (1989). Inoculations were done, at three different points, by forcing a 30′/2 gauge needle into the plumule, cotyledonary node, and adjacent regions and injecting about 30 mL of Agrobacterium cells at each injection point. The germination process of the Agrobacterium-infected seeds was continued by placing seeds on a sterile moistened paper towel and incubating at 26° C., in the dark, for about 4 h followed by transfer to soil for full development. Alternatively, the protocol described in Keshamma et al. (2008) was followed. Namely, the seedlings with just emerging plumule were infected by separating the cotyledons without damaging them such that the meristem is visible, and then pricked at the meristem with a sterile sewing needle and subsequently dunked in the culture of Agrobacterium for 60 min. Following infection, the seedlings were washed briefly with sterile water and later transferred to autoclaved soilrite.

BOMBARDMENT PDS or Other Bench Gene Guns Bombardment of Embryo Meristems

Bombardment of embryo meristem was done as follows: embryos in the coleoptilar or heart stage were disposed in the center of an embryo maturation media supplemented with osmoticum agents like described in Vain et al. (1993) 0-6 hours prior to bombardment. Particle preparation was done following a routine DNA precipitation with calcium chloride and spermidine. In the case of protein/RNA mix delivery it was done following the protocol described in Martin-Ortigosa et al. (2014) where the mix is dried or freeze dried along with the gold particles. 16-24 h after bombardment the embryos were moved to a maturation media without osmoticum in darkness.

Bombardment of Anthers

According to Twell (Twell et al. 1989), the anthers from greenhouse-grown plants were collected 1 d before pollen-shed, surface-sterilized in 10% Clorox for 10 min, and rinsed in sterile distilled water. The anthers were sliced transversely with a sterile razor blade, and 20 anther sections placed onto solid MSO medium in an area of 4 cm² with the anther locules exposed.

In another approach anthers were bombarded as described by Obert (Obert et al. 2008). For this protocol, spikes were harvested when microspores were in the mid-uninucleate stage of development. Two different pre-treatments were involved in our studies together with the use of non-pretreated material. For cold pre-treatment, collected spikes were stored on moistened filter paper in a cold room at 5° C. (Dedicova et al. 1999). After the pre-treatment of material (for 14 days in cold room), the specific parts of the spikes containing microspores at the appropriate exact stage were selected and used as a further experimental material, surface sterilised in 70% (v/v) alcohol and washed with sterile distilled water three times. For mannitol pre-treatment, suitable parts of the fresh spikes (containing microspores at the correct stage) were surface-sterilised in 70% alcohol and washed with sterile distilled water three times. Anthers were isolated in sterile conditions and placed in liquid media for pre-treatment (0.3 M mannitol or water) and stored in a cold room at 5° C. for 5 days. Anthers either freshly isolated or after pre-treatment were placed on the surface of cultivation media (FHG media Kasha et al. 2001) prior to bombardment. The bombardment set up was as folows: distance (macrocarrier—anthers in petri dish) 9 cm and pressure settings of 650, 900 and 1,100 p.s.i. Anther cultures were then cultured in a tissue culture growth cabinet at 26° C. in the dark.

In another approach, the protocol described by Touraev et al. (1997) was followed: To this end, unicellular microspores and midbicellular pollen grains were bombarded immediately after isolation in the respective culture medium. The suspension (0.7 ml) containing 5×10⁵ cells was distributed evenly on a sterile filter paper (Whatman No.1) and placed in a 10 cm Petri dish (Sterilin, UK). The helium-driven PDS-1000/He particle delivery system (Bio-Rad, USA) was used for biolistic transformation. Bombardment was performed essentially as described by Sanford et al. (1993). Plasmid DNA was precipitated onto gold particles (Bio-Rad, USA) with an average diameter of 1.1 μm. Each transformation event consisted of three bombardments. The bombarded microspores and midbicellular pollen grains were washed from the filter paper and incubated in the respective maturation medium.

Bombardment of Flowers

Flower bombardment can be conducted as described in Twell (Twell et al. 1989). For this approach, groups of 10 flowers with reflexed petals were bombarded intact with the cut pedicel suspended in distilled water.

Bombardment of Pollen

Pollen bombardment can be performed as described in Twell (Twell et al. 1989). Pollen have to be collected in sterile microfuge tubes from mature dehiscent flowers. Before bombardment, dry pollen samples are suspended in liquid MSO medium at a density of approximately 106 grains per mL. The pollen suspension (1 mL) was immediately pipetted onto the surface of a 9 cm Petri plate containing agar solidified MSO medium onto which sterile Whatman No. 1 filter paper overlayed with a nylon membrane (Genescreen, NEN) had been placed. Bombardment was performed within 60 min of placing plant materials onto MSO medium. Precipitation of plasmid DNA onto tungsten microprojectiles and bombardment was carried out as described in Klein et al. After bombardment, Petri plates or intact flowers in distilled water were incubated at 26° C. in the light.

In another approach, pollen bombardment was done as described in Horikawa et al. (1997). Namely, the mature pollen grains were collected from the extruding tassels. The following preparations for bombardment by a particle gun were performed quickly, since the viability of pollen diminished within a short time. The pollen was immersed in liquid MS medium containing 30 g/l saccharose (pH 5.8). The 4.0×10⁵ pollen grains in the 1 mL liquid medium were adsorbed on a surface of a piece of microfilter (pore size 0.45 μm, Fuji Film Co., Tokyo) by vacuum filtration. The microfilter was set on the 1% agar plate in a petri dish in preparation for the bombardment by a particle gun.

Pollination with the Treated Pollen

For pollination with the bombarded pollen the protocol described in (Touraev et al. 1997) was followed: Mature flowers just before anthesis with still closed anthers were emasculated one day before pollination. The in vitro matured pollen was washed several times in medium GK without quercetin and then transferred onto stigmas in a droplet of 3 μl. Stigmas were selected for pipet pollination which had produced a well developed stigmatic exudate. To prevent cross pollination, all the other flower buds in the climate chamber were removed every day before opening. Mature seed pods were collected after 3-4 weeks.

In another approach, the procedure described in Horikawa et al. (1997) was followed. For this, pollen was moved into the 1 mL liquid MS medium. The pollen was immediately pollinated by pipetting onto the silks of an ear covered with earbags, 3 days after silking. The pollination treatments were carried out for 20 ears. As a control, pollen bombarded with no DNA was pollinated.

Bombardment Helios Bombardment of Flowers

Bombardments of flowers or inflorescences were done using the hand held gun Helios from Bio-Rad according to the instructions of the manufacturer. Once the inflorescences or flowers were exposed 1 to 5 shots at 50-300 psi pressure were done. The exposed meristem was then covered and the inflorescence or flower was allowed to mature.

In another approach, the protocol described in (Gounaris et al. 2005) was followed: Flowers to be used as pollen receptors in pollination experiments were emasculated two days before the anticipated time of anther dehiscence. In the morning of the day when pollination was to be performed, donor flowers with intact stamens were collected 1-2 hours after dehiscence. Each donor flower was given 4-5 shots with the particle gun, all around it, while laying on a flat surface in a Petri dish and covered with a nylon mesh. The particle gun was operating under a helium pressure of 400 pounds per square inch (psi) and was equipped with a particle diffusion screen. The helium gas purity was grade 4.5 (99.994%). Each bombarded inflorescence was used to pollinate 15-20 emasculated receptor flowers. The pollinated flowers were allowed to develop and produce seeds.

Microinjection: DNA/RNA/Protein and Combinations Embryo Microinjection

For embryo microinjection the procedure described in Neuhaus (Neuhaus et al. 1987) was followed. Briefly, embryos were individually selected under optical control with a hand-drawn microcapillary connected to silicone tubing and transferred for microinjection to 2 μl droplets of the same medium positioned onto a coverslip (Spangenberg et al. 1986 a). Microinjection was performed by fixing the embryoids with a holding capillary and microinjecting into each cell. Exogenous DNA was injected as a 1:1 mixture of linearized (by cutting the plasmids outside of the inserted genes) and supercoiled molecules at 0.5 μg/μl in 50 mM NaCl, 50 mM Tris-HCl pH 7.8.

Microinjection of Agrobacterium in Shoot Meristems

The microinjection of shoot meristems was done as described by Sivakumar et al. (2014). Namely, 100 μL of the culture was taken in an insulin syringe and microinjected into the embryonic shoot apical meristem of germinated cotton seeds. The culture was microinjected (0.5-1.0 mm depth) one to five times to check the effect of number of microinjection in and around the embryonic shoot apical meristem. Excess bacterial culture was removed by blotting the infected seeds on sterile filter paper (Whatman No.1). The seeds were co-cultivated on ½ strength MS medium for two days in dark condition. After co-cultivation the seedlings were washed with Cefotaxime (200 mg/L) and transferred to selection medium containing Cefotaxime and Hygromycin-B antibiotics.

Microinjection of DNA in Shoot Meristems

Microinjection was performed following the protocol described in Lusardi et al. (1994). Mature, dried seeds were washed with absolute ethanol for 20 sec followed by sterilization with commercial bleach (2.5% NaClO) supplemented with 0.01% Tween 80, for 20 min with shaking. Seeds were then rinsed four to five times with sterile distilled water. Germination was induced by incubating the seeds in a 9 cm petri dish between filter papers soaked with sterile distilled water, at 27° C., in darkness, for 3-4 days. During this time the shoot broke through the kernel teguments and reached 0.8-1.0 cm in length. At this stage the shoot was removed from the seed at the level of the scutellar node. Under the stereo microscope the coleoptile and the five or six embryonic leaves present at this developmental stage were removed. After dissection of the embryonic leaves, the uncovered apex surrounded by two leaf primordia at different developmental stages was exposed. The isolated apices were cultivated in 9 cm petri dishes in MS medium (Murashige and Skoog, 1962) supplemented with 2% sucrose and solidified with 0.8% Difco Bacto-Agar (Difco Lab., Detroit) with a 27° C./22° C. temperature regime and a 16/8 h light/dark photoperiod. Within 10 days a normal plantlet developed and during the next 15-20 days it reached a size sufficient for transfer to pots and to the greenhouse. For the microinjection, the plasmids for injection were dissolved at a concentration of 0.1-0.5 μg/μl in injection buffer (10 mM Tris-HCl and 0.1 M EDTA, pH 7.5). The injection buffer was filtered through a 0.2 μm disposable filter unit (Schleicher and Schuell, Germany) to sterilize the solution and to avoid particle contaminations. All injections were done under sterile conditions. The isolated shoot meristems from maize were transferred into 9 cm petri dishes containing MS medium supplemented with 2% sucrose and solidified with 0.8% Difco Bacto Agar. The apices were oriented on the medium so that the apical dome was clearly visible. The cells of the L2 layers of the meristems were injected using an Embryo Splitter System from Research Instruments (UK) equipped with a high magnification stereo microscope (up to 200×magnification; SV 8, Zeiss, Germany). In several experiments co-injection of FITC-dextran was used to identify the injected cell (Neuhaus et al., 1993; Schnorf et al., 1991).The mechanical micromanipulator of the system carries an injection capillary (tip diameter less than 1 μm) connected to a microinjector (Eppendorf 5242 Microinjector) which delivers constant volumes of about 3 pl into the cells (Neuhaus et al., 1986; 1987; Schnorf et al., 1991). For stability and movement of the apices during injection the second manipulator of the Embryo Splitter System was used. For this purpose this manipulator was also equipped with a microneedle to move and fix the apical meristems to be injected in the correct position.

Whiskers

Whisker mediated delivery was applied to the different meristems as described in Frame et al. (1994). Namely, 40 microlitres of 5% whisker suspension and 25 μl of plasmid DNA (1 μg/μl) were added to the exposed tissues. Tube contents were first finger tapped to mix and then placed either upright in a multiple sample head on a Vortex Genie II vortex mixer (Scientific Industries Inc., Bohemia, N.Y.) or horizontally in the holder of a Mixomat dental amalgam mixer (Degussa Canada Lt.d, Burlington, Ontario). Transformation was carried out by mixing at full speed for 60 sec (Vortex Genie II) or shaking at fixed speed for 1 sec (Mixomat). Alternatively, whiskers were loaded in the pipette of a micromanipulator along with DNA/RNA or protein mix and were macro-injected to meristematic tissues.

Cell Penetrating Peptides: DNA/RNA/Protein and Combinations Mixing Cell Penetrating Peptides and Cas9 Protein and gRNA

For applying a cell penetrating peptide approach, a protocol adapted from Ramakrishna (Ramakrishna et al. 2014) was used. One day after plating, target cells were washed with Opti-MEM and treated with Cas9-m9R and sgRNA:9R either sequentially or simultaneously. The sgRNA:9R complex was formed by incubation of 10 mg of sgRNA and 30-50 mg of 9R peptide in 250 mL (for sequential treatment) or 100 mL (for simultaneous treatment) of Opti-MEM medium at room temperature for 30 min.

Embryo

Tat peptides (Tat, Tat2, M-Tat) were employed for delivery of GUS enzyme in wheat embryos. Tat peptide and GUS enzyme were first prepared in separate microcentrifuge tubes. Nonlabeled Tat peptide (4 μg) was added to sterile water (with the final volume made up to 100 μL). Similarly, 1 μg of GUS enzyme (Sigma Aldrich) was added to sterile water to give a final volume of 100 μL. The contents of the two tubes were mixed together, giving a 4:1 peptide:protein ratio in the mixture. The mixture was incubated for 1 h at room temperature and then added to the isolated immature embryos (in a 2 mL microcentrifuge tube) in the presence or absence of permeabilizing agent toluene/ethanol (1:40, v/v with the total volume of the peptide:protein mixture). After 1 h of incubation at room temperature, embryos were washed twice with the permeabilization buffer and subjected to trypsin treatment (1:1 (v/v) with permeabilization buffer) for 5 min at room temperature. The embryos were washed twice with permeabilization buffer followed by GUS histochemical analysis of the embryos. For delivery of 1 μg of GUS enzyme by the Chariot protein transduction kit (Active Motif, Carlsbad, Calif., USA), the manufacturer's protocol was followed. Permeabilized and nonpermeabilized embryos were incubated with Chariot—GUS complex for 1 h. All post-incubation steps were the same as that described for Tat peptides.

Transformation of Microspores

Transformation of microspores using cell penentrating peptides was done according to Shim et al. (2012). Briefly, microspore extraction was conducted according to Eudes and Amundsen (2005), and all steps of microspore isolation were carried out using NPB-99 liquid medium (Zheng et al. 2001; Eudes and Amundsen 2005). After washing of microspores with NPB-99, 2-3 ml microspore solution was layered on 2-3 ml of 30% Percoll solution containing 400 mM mannitol and 10 mM MES, pH 7.0. Microspores were centrifuged for 5 min at 100×g at 4° C. The cells that formed a band at the Percoll/NPB-99 interface were transferred to a new 15-mL centrifuge tube, diluted to 15 ml with NPB-99, and then centrifuged again. The supernatant was decanted and the microspores were resuspended in about 1 ml of NPB-99 medium. The microspore concentration was determined using a hemocytometer and adjusted to 2.5×10⁵cells/ml. Five treatments, including the control, were applied to microspore suspensions from the same extraction, as follows: T1, control treatment comprised of 200 μl of sterile water; T2, 1 μg of dsDNA diluted in 100 μl sterile water was added to 4 μg of Tat2 diluted in 100 μl sterile water and gently mixed together, resulting in a 1:4 ratio of dsDNA to Tat2 (dsDNATat2); T3, 1 μg of dsDNA diluted in 100 μl sterile water and 6 μl of Chariot (Active Motif, Carlsbad, Calif.) diluted in 100 μl sterile water were mixed together (dsDNA-Pep1); T4, 4 μg of RecA (MJS Biolynx, Brockville, Canada; #UB70028Z) in 50 μl sterile water and 1 μg of dsDNA in 50 μl of sterile water were mixed together for 15 min and 6 μl of Chariot in 100 μl sterile water was added to the dsDNA-RecA solution for a final volume of 200 μl in a 2-ml microcentrifuge tube (dsDNA-RecA-Pep1); T5, 4 μg of RecA in 50 μl sterile water and 1 μg of dsDNA in 50 μl of sterile water were mixed together for 15 min. Four micrograms of Tat2 in 100 μl sterile water was pipetted to the dsDNA-RecA solution for a final volume of 200 μl in a 2-ml microcentrifuge tube (dsDNA-RecA-Tat2). Following incubation for 15 min at room temperature (RT), 5 pl of Lipofectamine (Invitrogen, Carlsbad, Calif.; #11668-019) was added to all treatments and left to stand for another 5 min at RT. The mixtures were then immediately added to 50,000 pelleted microspores in 2 ml microcentrifuge tubes and incubated for 15 min. Then, 100 μl of NPB-99 was added per tube and incubated at RT for 45 min. Transfected microspores were pelleted, the supernatant was removed, and cells were washed twice with NPB-99. Then, 1 ml NPB-99 was added per tube of microspores, gently mixed, and aliquots of 500 μl were pipetted into 35-mm Petri dishes containing 3 ml NPB-99+10% Ficoll (Sigma, St. Louis, Mo.; F4375; NPB-99-10F), and 100 mg/L of the antibiotic cefotaxime (Sigma; #C7039).

Electroporation Pollen Transformation

Pollen transformation through electroporation was done following the protocol described by Shi et al. (1996). Briefly, mature pollen, germinating pollen or pollen without exine coat was electroporated with a field strength of 750-1250 V/cm with a time constant pulse of 13 ms.

Sonication Pollen Ultrasonication

Pollen transformation through ultrasonication was done as described by Wang et al. (2000). Namely, 0.3 g of fresh pollens were collected in the morning, and mixed with about 10 μg of the plasmid DNA in 20 mL of solution with 5% sucrose. The solution was treated with ultrasonication before and after adding the plasmid DNA. By using a JY92-II Ultrasonicator from Ningbo Xinzi Scientific Instrument Institute the parameters for sonication treatment were: sonic intensity of 300 W, treatments for 8 times each for 5 s and 10 s interval. Then, the treated pollens were pollinated on clipped maize silks.

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1-15. (canceled)
 16. A method for the in planta transformation of a plant or plant material with a genetic construct of interest comprising the following steps: providing a plant comprising at least one meristematic cell of a meristematic tissue of an immature inflorescence that is able to differentiate into a gamete of a pollen or of an ovule; (ii) exposing the at least one meristematic cell of the meristematic tissue of the immature inflorescence; (iii) providing a genetic construct of interest; and (iv) transforming the at least one meristematic cell of the meristematic tissue of the immature inflorescence by introducing the genetic construct of interest under suitable conditions to allow the functional integration of the genetic construct of interest into the at least one meristematic cell of the meristematic tissue of the immature inflorescence.
 17. The method according to claim 16, wherein the transformation is performed in vitro culture free.
 18. The method according to claim 16, wherein the functional integration of the genetic construct of interest into the at least one meristematic cell of the meristematic tissue of the immature inflorescence of step (iv) is performed as (a) a stable integration so that the integrated genetic construct of interest, or a part thereof, is heritable to a progeny as transgene, optionally wherein a transformed gamete of the pollen or of the ovule is generated from the at least one transformed meristematic cell and the transformed gamete of the pollen or of the ovule is fused to a zygote from which the progeny comprising the genetic construct of interest is developed; or (b) a transient introduction allowing a targeted genetic manipulation of the at least one meristematic cell of the meristematic tissue of the immature inflorescence through the genetic construct of interest or products thereof, wherein the targeted genetic manipulation but not the genetic construct of interest, or a part thereof, is heritable to a progeny, optionally wherein a genetic manipulated gamete of the pollen or of the ovule is generated from the at least one transformed meristematic cell and the genetic manipulated gamete of the pollen or of the ovule is fused to a zygote from which the progeny comprising the targeted genetic manipulation is developed.
 19. The method according to claim 16, wherein the method further comprises (v) comprising determining the functional integration of the genetic construct of interest.
 20. The method according to claim 16, wherein the introduction of the genetic construct of interest according to step (iv) is conducted using a means selected from the group consisting of a device suitable for particle bombardment, transformation, microinjection, electroporation, whisker technology, transfection, or a combination of one or more of particle bombardment, transformation, microinjection, electroporation, whisker technology and transfection.
 21. The method according to claim 20, wherein the device suitable for particle bombardment is a gene gun, transformation is performed with Agrobacterium spp. or a viral vector, or the whisker technology is silicon carbide whisker technology.
 22. The method according to claim 16, wherein step (ii) comprises the production of an artificially introduced window in the region, optionally wherein the meristematic tissue of the immature inflorescence is located in the shoot axis.
 23. The method according to claim 22, the meristematic tissue of the immature inflorescence is located in the shoot axis and in the axil, halm, culm or stem of the plant.
 24. The method according to claim 16, wherein the plant or the plant material is or is part of a monocotyledonous plant, or is or is part of a dicotyledonous plant.
 25. The method according to claim 16, wherein the plant or the plant material is or is part of a plant from a species selected from the group consisting of Hordeum vulgare, Hordeum bulbusom, Sorghum bicolor, Saccharum officinarium, Zea mays, Setaria italica, Oryza minuta, Oryza sativa, Oryza australiensis, Oryza alta, Triticum aestivum, Triticale, Secale cereale, Malus domestica, Brachypodium distachyon, Hordeum marinum, Aegilops tauschii, Daucus glochidiatus, Beta vulgaris, Daucus pusillus, Daucus muricatus, Daucus carota, Eucalyptus grandis, Nicotiana sylvestris, Nicotiana tomentosiformis, Nicotiana tabacum, Solanum lycopersicum, Solanum tuberosum, Coffea canephora, Vitis vinifera, Erythrante guttata, Genlisea aurea, Cucumis sativus, Morus notabilis, Arabidopsis arenosa, Arabidopsis lyrata, Arabidopsis thaliana, Crucihimalaya himalaica, Crucihimalaya wallichii, Cardamine flexuosa, Lepidium virginicum, Capsella bursa pastoris, Olmarabidopsis pumila, Arabis hirsute, Brassica napus, Brassica oeleracia, Brassica rapa, Raphanus sativus, Brassica juncea, Brassica nigra, Eruca vesicaria subsp. sativa, Citrus sinensis, Jatropha curcas, Populus trichocarpa, Medicago truncatula, Cicer yamashitae, Cicer bijugum, Cicer arietinum, Cicer reticulatum, Cicer judaicum, Cajanus cajanifolius, Cajanus scarabaeoides, Phaseolus vulgaris, Glycine max, Gossypium sp., Astragalus sinicus, Lotus japonicas, Torenia fournieri, Allium cepa, Allium fistulosum, Allium sativum, Helianthus annuus, Helianthus tuberosus and Allium tuberosum.
 26. The method according to claim 16, wherein the plant or the plant material is or is part of a plant from any variety or subspecies belonging to a species selected from the group consisting of Hordeum vulgare, Hordeum bulbusom, Sorghum bicolor, Saccharum officinarium, Zea mays, Setaria italica, Oryza minuta, Oryza sativa, Oryza australiensis, Oryza alta, Triticum aestivum, Triticale, Secale cereale, Malus domestica, Brachypodium distachyon, Hordeum marinum, Aegilops tauschii, Daucus glochidiatus, Beta vulgaris, Daucus pusillus, Daucus muricatus, Daucus carota, Eucalyptus grandis, Nicotiana sylvestris, Nicotiana tomentosiformis, Nicotiana tabacum, Solanum lycopersicum, Solanum tuberosum, Coffea canephora, Vitis vinifera, Erythrante guttata, Genlisea aurea, Cucumis sativus, Morus notabilis, Arabidopsis arenosa, Arabidopsis lyrata, Arabidopsis thaliana, Crucihimalaya himalaica, Crucihimalaya wallichii, Cardamine flexuosa, Lepidium virginicum, Capsella bursa pastoris, Olmarabidopsis pumila, Arabis hirsute, Brassica napus, Brassica oeleracia, Brassica rapa, Raphanus sativus, Brassica juncea, Brassica nigra, Eruca vesicaria subsp. sativa, Citrus sinensis, Jatropha curcas, Populus trichocarpa, Medicago truncatula, Cicer yamashitae, Cicer bijugum, Cicer arietinum, Cicer reticulatum, Cicer judaicum, Cajanus cajanifolius, Cajanus scarabaeoides, Phaseolus vulgaris, Glycine max, Gossypium sp., Astragalus sinicus, Lotus japonicas, Torenia fournieri, Allium cepa, Allium fistulosum, Allium sativum, Helianthus annuus, Helianthus tuberosus and Allium tuberosum.
 27. The method according to claim 16, wherein the immature inflorescence is a male inflorescence (tassel) of a Zea mays plant, or wherein the immature inflorescence is the inflorescence of a Triticum aestivum plant.
 28. The method according to claim 27, wherein the introduction of the genetic construct of interest as defined in step (iv) is performed at a developmental stage, either during the stamen initiation process or before spikelets are formed on the male inflorescence.
 29. A method for manufacturing a transgenic plant comprising the following steps: (I) providing an in planta transformed plant or plant material transformed by a method according to claim 16; (II) cultivating the plant or plant material under suitable conditions until the developmental stage of maturity of the inflorescence is achieved, (III) allowing the transformed gamete of the pollen or of the ovule generated from the transformed at least one meristematic cell to fuse to a zygote comprising the genetic construct of interest as transgene; and (IV) developing the transgenic plant from the zygote.
 30. A method for manufacturing a genetically manipulated plant comprising the following steps: (I) providing an in planta transformed plant or plant material transformed by a method according to claim 16; (II) cultivating the plant or plant material under suitable conditions until the developmental stage of maturity of the inflorescence is achieved; (III) allowing the genetically manipulated gamete of the pollen or of the ovule generated from the transformed at least one meristematic cell to fuse to a zygote comprising the targeted genetic manipulation; and (IV) developing the genetically manipulated plant from the zygote.
 31. A plant manufactured by the method of claim 27 or plant cells, a plant material, or derivatives, or a progeny thereof
 32. A plant comprising an artificially introduced window in a region where a meristematic tissue of an immature inflorescence is located, optionally wherein the plant is a Zea mays plant, optionally wherein the meristematic tissue of the immature inflorescence is located in the shoot axis of the plant, such as in the axil, halm, culm or stem of the plant, and optionally wherein the plant is a plant in planta transformed by a method according to claim
 16. 33. A method for the manufacture of a transgenic or genetically manipulated plant, plant cell or plant material comprising utilizing a plant according to claim
 31. 34. A method for the manufacture of a transgenic or genetically manipulated plant, plant cell or plant material comprising utilizing a plant in planta transformed by a method according to claim
 16. 